Polynucleotides encoding IL-17/IL-23 bispecific, IL-17A/F and IL-23p19 antibodies

ABSTRACT

The present invention relates to antagonizing the activity of IL-17A, IL-17F and IL-23 using bispecific antibodies that comprise a binding entity that is cross-reactive for IL-17A and IL-17F and a binding entity that binds IL-23p19. The present invention relates to novel bispecific antibody formats and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/898,544, filed May 21, 2013, now U.S. Pat. No. 8,945,553, whichclaims the benefit of U.S. Patent Application Ser. No. 61/787,890, filedMar. 15, 2013, U.S. Patent Application Ser. No. 61/784,600, filed Mar.14, 2013, and U.S. Patent Application Ser. No. 61/650,286, filed May 22,2012, all of which are herein incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

Incorporated herein by reference in its entirety is a Sequence Listingentitled, “20130521_SEQ_11921USNP.txt”, comprising SEQ ID NO:1 throughSEQ ID NO:126, which include nucleic acid and/or amino acid sequencesdisclosed herein. The Sequence Listing has been submitted herein inASCII text format via EFS, and thus constitutes both the paper andcomputer readable form thereof. The Sequence Listing was first createdusing PatentIn on May 21, 2013, and is 421 KB.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the induction of immune cellproliferation, development, differentiation, and/or migration, as wellas the regulation of the growth and differentiation of many cell types(see, for example, Arai et al., Annu. Rev. Biochem. 59:783 (1990);Mosmann, Curr. Opin. Immunol. 3:311 (1991); Paul et al., Cell, 76:241(1994)). Cytokine-induced immune functions can also include aninflammatory response, characterized by a systemic or local accumulationof immune cells. Although they do have host-protective effects, theseimmune responses can produce pathological consequences when the responseinvolves excessive and/or chronic inflammation, as in autoimmunedisorders (such as multiple sclerosis) and cancer/neoplastic diseases(Oppenheim et al., eds., Cytokine Reference, Academic Press, San Diego,Calif. (2001); von Andrian et al., New Engl. J. Med., 343:1020 (2000);Davidson et al., New Engl. J. Med., 345:340 (2001); Lu et al., Mol.Cancer. Res., 4:221 (2006); Dalgleish et al., Cancer Treat Res., 130:1(2006)).

IL-17A, IL-17F and IL-23 are cytokines involved in inflammation. IL-17Ainduces the production of inflammatory cytokines such as IL-1β, TNF-α,IL-6, and IL-23 by synovial fibroblasts, monocytes, and macrophages, allof which promote inflammation and Th17 development. IL-17A also inducesan array of chemokines, including CXCL-1, CXCL-2, CXCL-5, CXCL-8, CCL-2,and CCL-20, leading to recruitment of T cells, B cells, monocytes, andneutrophils. Lundy, S. K., Arthritis Res. Ther., 9:202 (2007). IL-17Fshares the greatest homology (55%) with IL-17A and is also aproinflammatory cytokine. Both IL-17A and IL-17F are produced by Th17cells, whereas the other IL-17 family members, IL-17B, IL-17C, andIL-17D, are produced by non-T cell sources. IL-17A and IL-17F can existas IL-17A homodimers and IL-17F homodimers or as IL-17A/F heterodimers.Liang, S. C. et al., J. Immunol., 179:7791-7799 (2007). IL-17A isincreased in rheumatoid arthritis sera and synovial fluid, and ispresent in the T-cell rich areas of the synovium. Shahrara, S.,Arthritis Res. Ther., 10:R93 (2005). IL-17A can also orchestrate boneand cartilage damage. An effective blockade of IL-17 will need toneutralize IL-17A homodimers, IL-17F homodimers and IL-17A/Fheterodimers.

IL-23 is a type-1 heterodimer, comprising a 19 kilodalton (kD) fourfoldhelical core α subunit (IL-23p19), disulfide linked to an additional 40kD distinct β subunit (IL-12p40). IL-23 is a key cytokine in bridgingthe innate and adaptive arms of the immune response; it is producedearly in response to an antigen challenge, and is essential for drivingearly local immune responses. Furthermore, IL-23 plays a central role inthe activation of NK cells, the enhancement of T cell proliferation andthe regulation of antibody production. IL-23 also regulatespro-inflammatory cytokines (e.g., IFN-γ), which are important incell-mediated immunity against intracellular pathogens. Recent reportshave indicated that in humans increased amounts of IL-23 have beenassociated with several autoimmune diseases including rheumatoidarthritis (RA), Lyme arthritis, inflammatory bowel disease (IBD),Crohn's disease (CD), psoriasis and multiple sclerosis (MS). IL-23p19knock-out mice were resistant to autoimmune encephalomyelitis (EAE),collagen-induced arthritis (CIA) and central nervous system autoimmuneinduction. IL-23 is not essential for the development of human Th17cells, but appears to be required for their survival and/or expansion.Paradowska-Gorycka, A., Scandinavian Journal of Immunology, 71:134-145(2010). Genetic studies revealed an association between IL-23 receptorgenes and susceptibility to several autoimmune diseases including CD, RAand Graves' ophthalmopathy. The IL-23-Th17 axis is crucial to autoimmunedisease development. Leng et al., Archives of Medical Research,41:221-225 (2010).

The demonstrated activities of IL-17A, IL-17F and IL-23p19 in mediatingand promoting several autoimmune diseases illustrate the clinicalpotential of, and need for, molecules which can antagonize thesetargets. The present invention, as set forth herein, meets these andother needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a whole antibody and its modularcomponents.

FIG. 2 depicts a model of a bispecific antibody designated biAbFabLwhich contains a whole antibody with a C-terminal Fab unit of the secondarm of the bispecific antibody attached via a linker, and which utilizesa common light chain.

FIG. 3 depicts a model of a bispecific antibody designated taFab whichcontains a whole antibody with an N-terminal Fab unit of the second armof the bispecific antibody attached via a linker. As with the heavychain portion, there are two light chains for each arm of the bispecificattached via a linker.

FIG. 4 depicts a model of a bispecific antibody designated HeterodimericFc, that resembles a traditional antibody, however, contains twodifferent heavy chains which associate through an electrostaticcomplementarity association in the C region. The Heterodimeric Fcutilizes a common light chain.

FIG. 5 depicts a model of a bispecific antibody designated VCVFc whichcontains a whole antibody with a Fv unit of the second arm of thebispecific antibody inserted between the Fab region and the hinge vialinkers.

FIG. 6 depicts a model of a bispecific antibody designated VCDFc whichcontains a whole antibody with a single domain antibody for the secondarm of the bispecific antibody inserted between the Fab region and thehinge via linkers.

FIG. 7 illustrates the ELISA results showing strong antibody binding toIL-23p19 and lack of cross reactivity to IL-12.

FIG. 8 illustrates the potent neutralization of IL-23 signaling asobserved in the kit225 assay.

FIG. 9 shows the Biacore results of 7B7, STELARA® (ustekinumab, ananti-IL-23p40 antibody) and human IL-23 receptor ability to bind thevarious IL-23p19 alanine shaved mutants, wild-type and purifiedwild-type L-23p19 (positive control) and a negative control. 7B7 mAbbinding is shown in the left column, STELARA® is shown in the centercolumn and hIL-23R-Fc binding is shown in the right column. The fourmutants shown with the star were chosen for scale-up to confirm theseresults.

FIG. 10 shows a Biacore kinetic analysis of IL-23p19 antibody binding tothe IL-23 alanine mutants.

FIG. 11 schematically illustrates the process used to identify andselect the 7B7 antibody (anti-IL-23p19).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one embodiment, bispecific antibodiescomprising an IL-17A/F binding entity that binds to IL-17A and IL-17Fand an IL-23 binding entity that binds to IL-23 via p19. The inventionalso includes isolated nucleic acids encoding the heavy chains and lightchains of the bispecific antibodies of the invention, as well as vectorscomprising the nucleic acids, host cells comprising the vectors, andmethods of making and using the bispecific antibodies.

In other embodiments, the present invention provides compositionscomprising the bispecific antibodies and kits comprising the bispecificantibodies, as well as articles of manufacture comprising the bispecificantibodies.

The bispecific antibodies of the present invention are useful for theinhibition of proinflammatory cytokines, e.g., IL-17A, IL-17F andIL-23p19. The antibodies can be used to reduce, limit, neutralize, orblock the proinflammatory effects of the IL-17A homodimer, the IL-17Fhomodimer, or the IL-17A/F heterodimer. Likewise, the antibodies can beused to reduce, limit, neutralize, or block the pro-cancerous effects ofthe IL-17A homodimer, the IL-17F homodimer, or the IL-17A/F heterodimer.In such cases, the anti-IL-23p19 portion of the antibody is used toreduce, limit, neutralize, or block production of new T cells that wouldproduce IL-17A and/or IL-17F, including homodimers and heterodimers. Thebispecific antibodies described herein can be used to treat inflammatorydisorders and autoimmune diseases, such as multiple sclerosis,inflammatory bowel disease, psoriasis, systemic sclerosis, systemiclupus erythematosus, antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis (AAV) and giant cell arteritis. Thebispecific antibodies described herein can also be used to treat cancer,including angiogenesis. For instance, the bispecific antibodies asdescribed herein can be used to treat multiple-myeloma-induced lyticbone disease (Sotomayor, E. M., Blood, 116(18):3380-3382 (2010)).

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a”, “an”, “the”, and “at least one” areused interchangeably and mean one or more than one.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.Thus, as used herein, the term “antibody” or “antibody peptide(s)”refers to an intact antibody, or an antigen-binding fragment thereofthat competes with the intact antibody for specific binding and includeschimeric, humanized, fully human, and bispecific antibodies. In certainembodiments, antigen-binding fragments are produced, for example, byrecombinant DNA techniques. In additional embodiments, antigen-bindingfragments are produced by enzymatic or chemical cleavage of intactantibodies. Antigen-binding fragments include, but are not limited to,Fab, Fab′, F(ab)², F(ab′)², Fv, and single-chain antibodies.

The term “isolated antibody” as used herein refers to an antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule.

The term “bind(ing) of a polypeptide” includes, but is not limited to,the binding of a ligand polypeptide of the present invention to areceptor; the binding of a receptor polypeptide of the present inventionto a ligand; the binding of an antibody of the present invention to anantigen or epitope; the binding of an antigen or epitope of the presentinvention to an antibody; the binding of an antibody of the presentinvention to an anti-idiotypic antibody; the binding of ananti-idiotypic antibody of the present invention to a ligand; thebinding of an anti-idiotypic antibody of the present invention to areceptor; the binding of an anti-anti-idiotypic antibody of the presentinvention to a ligand, receptor or antibody, etc.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990);Kostelny et al., J. Immunol., 148:1547-1553 (1992).

As used herein, the term “epitope” refers to the portion of an antigento which an antibody specifically binds. Thus, the term “epitope”includes any protein determinant capable of specific binding to animmunoglobulin or T-cell receptor. Epitopic determinants usually consistof chemically active surface groupings of molecules such as amino acidsor sugar side chains and usually have specific three dimensionalstructural characteristics, as well as specific charge characteristics.More specifically, the term “IL-17 epitope”, “IL-23 epitope” and/or“IL-23/p19 epitope” as used herein refers to a portion of thecorresponding polypeptide having antigenic or immunogenic activity in ananimal, preferably in a mammal, and most preferably in a mouse or ahuman. An epitope having immunogenic activity is a portion of, forexample, an IL-17A or IL-17F or IL-23/p19 polypeptide that elicits anantibody response in an animal. An epitope having antigenic activity isa portion of, for example, an IL-17A or IL-17F or IL-23/p19 polypeptideto which an antibody immunospecifically binds as determined by anymethod well known in the art, for example, by immunoassays, proteasedigest, crystallography or H/D-Exchange. Antigenic epitopes need notnecessarily be immunogenic. Such epitopes can be linear in nature or canbe a discontinuous epitope. Thus, as used herein, the term“conformational epitope” refers to a discontinuous epitope formed by aspatial relationship between amino acids of an antigen other than anunbroken series of amino acids.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or about 214amino acids) are encoded by a variable region gene at the NH2-terminus(about 110 amino acids) and a kappa or lambda constant region gene atthe COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50Kd or about 446 amino acids), are similarly encoded by a variable regiongene (about 116 amino acids) and one of the other aforementionedconstant region genes (about 330 amino acids). Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG (such as IgG1, IgG2, IgG3 and IgG4), IgM, IgA,IgD and IgE, respectively. Within light and heavy chains, the variableand constant regions are joined by a “J” region of about 12 or moreamino acids, with the heavy chain also including a “D” region of about10 more amino acids. (See generally, Fundamental Immunology (Paul, W.,ed., 2nd Edition, Raven Press, NY (1989)), Chapter 7 (incorporated byreference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Edition, Public Health Service, National Institutes ofHealth, Bethesda, Md. (1991)) and/or those residues from a“hypervariable loop” (Chothia et al., J. Mol. Biol. 196: 901-917 (1987))(both of which are incorporated herein by reference). “Framework Region”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. Thus, a “human framework region” is aframework region that is substantially identical (about 85% or more,usually 90-95% or more) to the framework region of a naturally occurringhuman immunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen. Accordingly, theterm “humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. Constant regions need not bepresent, but if they are, they must be substantially identical to humanimmunoglobulin constant regions, i.e., at least about 85-90%, preferablyabout 95% or more identical. Hence, all parts of a humanizedimmunoglobulin, except possibly the CDR's, are substantially identicalto corresponding parts of natural human immunoglobulin sequences.Further, one or more residues in the human framework region may be backmutated to the parental sequence to retain optimal antigen-bindingaffinity and specificity. In this way, certain framework residues fromthe non-human parent antibody are retained in the humanized antibody inorder to retain the binding properties of the parent antibody whileminimizing its immunogenicity. The term “human framework region” as usedherein includes regions with such back mutations. A “humanized antibody”is an antibody comprising a humanized light chain and a humanized heavychain immunoglobulin. For example, a humanized antibody would notencompass a typical chimeric antibody as defined above, e.g., becausethe entire variable region of a chimeric antibody is non-human.

The term “humanized” immunoglobulin refers to an immunoglobulincomprising a human framework region and one or more CDR's from anon-human, e.g., mouse, rat or rabbit, immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's andpossibly a few back-mutated amino acid residues in the framework region(e.g., 1-15 residues), are substantially identical to correspondingparts of natural human immunoglobulin sequences. A “humanized antibody”is an antibody comprising a humanized light chain and a humanized heavychain immunoglobulin. For example, a humanized antibody would notencompass a typical chimeric antibody as defined above, e.g., becausethe entire variable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes an antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable and/or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

A “Fv fragment” contains the variable regions from both heavy and lightchains but lacks the constant regions.

A “single domain antibody” is an antibody fragment consisting of asingle domain Fv unit, e.g., V_(H) or V_(L). Like a whole antibody, itis able to bind selectively to a specific antigen. With a molecularweight of only 12-15 kDa, single-domain antibodies are much smaller thancommon antibodies (150-160 kDa) which are composed of two heavy proteinchains and two light chains, and even smaller than Fab fragments (˜50kDa, one light chain and half a heavy chain) and single-chain variablefragments (˜25 kDa, two variable domains, one from a light and one froma heavy chain). The first single-domain antibodies were engineered fromheavy-chain antibodies found in camelids. Although most research intosingle-domain antibodies is currently based on heavy chain variabledomains, light chain variable domains and nanobodies derived from lightchains have also been shown to bind specifically to target epitopes.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids”, whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol., 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol., 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem., 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem., 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr., 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th Edition, The Benjamin/Cummings Publishing Company, Inc.(1987), and Lemaigre et al., Biochem. J., 303:1 (1994)). If a promoteris an inducible promoter, then the rate of transcription increases inresponse to an inducing agent. In contrast, the rate of transcription isnot regulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific”,“tissue-specific”, or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces anantagonist of the present invention from an expression vector. Incontrast, such an antagonist can be produced by a cell that is a“natural source” of said antagonist, and that lacks an expressionvector.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a IL-17RApolypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of IL-17Ausing affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apolyhistidine tract, protein A (Nilsson et al., EMBO J., 4:1075 (1985);Nilsson et al., Methods Enzymol., 198:3 (1991)), glutathione Stransferase (Smith et al., Gene, 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA, 82:7952 (1985)),substance P, FLAG® peptide (Hopp et al., Biotechnology, 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification, 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

An “IL-17A binding entity” is a binding entity, such as an antibody,that specifically binds to IL-17A in its homodimeric form(IL-17A/IL-17A) and in its heterodimeric form (IL-17A/IL-17F).

An “IL-17F binding entity” is a binding entity, such as an antibody,that specifically binds to IL-17F in its homodimeric form(IL-17F/IL-17F) and in its heterodimeric form (IL-17A/IL-17F).

An “IL-17A/F binding entity” is a binding entity, such as an antibody,that specifically binds to IL-17A and IL-17F by recognizing and bindingto the same or similar epitope, e.g., continuous or discontinuousepitope, shared by IL-17A and IL-17F. The IL-17A/F binding entity bindsto the IL-17A homodimer, IL-17F homodimer and IL-17A/IL-17F heterodimer.

In one embodiment, the present invention provides bispecific antibodies,antibodies and antigen-binding fragments thereof. The bispecificantibodies of the invention comprise an IL-17A binding entity that bindsto IL-17A and an IL-23 binding entity that binds to IL-23 via p19. Inanother aspect, the bispecific antibodies of the invention comprise anIL-17F binding entity that binds to IL-17F and a IL-23 binding entitythat binds to IL-23 via p19. In another aspect, the bispecificantibodies of the invention comprise an IL-17A/F binding entity thatbinds to IL-17A and IL-17F and a IL-23 binding entity that binds toIL-23 via p19. The binding entity that binds to IL-23 via p19 isreferred to hereinafter as a binding entity that binds to IL-23 or an“IL-23 binding entity”. The polynucleotide sequence of the human IL-17Ais shown in SEQ ID NO:1 and the corresponding polypeptide sequence isshown in SEQ ID NO:2. The signal sequence of the IL-17A polypeptide isamino acid residues 1-23 of SEQ ID NO:2. Thus, amino acid residues24-155 of SEQ ID NO:2 constitute the mature IL-17A polypeptide.Antibodies (and antigen-binding fragments thereof) and bispecificantibodies disclosed herein that bind to IL-17A bind to the matureIL-17A polypeptide (amino acid residues 24-155 of SEQ ID NO:2). Thepolynucleotide sequence of the human IL-17F is shown in SEQ ID NO:3 andthe corresponding polypeptide sequence is shown in SEQ ID NO:4. Thesignal sequence of the IL-17F polypeptide is amino acid residues 1-30 ofSEQ ID NO:4. Thus, amino acid residues 31-163 of SEQ ID NO:4 constitutethe mature IL-17F polypeptide. Antibodies (and antigen-binding fragmentsthereof) and bispecific antibodies disclosed herein that bind to IL-17Fbind to the mature IL-17F polypeptide (amino acid residues 31-163 of SEQID NO:4). The polynucleotide sequence of the human p19 subunit of IL-23is shown in SEQ ID NO:5 and the corresponding polypeptide sequence isshown in SEQ ID NO:6. The signal sequence of the IL-23p19 polypeptide isamino acid residues 1-19 of SEQ ID NO:6. Thus, amino acid residues20-189 of SEQ ID NO:6 constitute the mature IL-23p19 polypeptide.Antibodies (and antigen-binding fragments thereof) and bispecificantibodies disclosed herein that bind to IL-23p19 bind to the matureIL-23p19 polypeptide (amino acid residues 20-189 of SEQ ID NO:6).

In one aspect of the invention, the IL-17A/F binding entity comprises anantibody, i.e., two pairs of immunoglobulin chains, each pair having onelight and one heavy chain, and the IL-23 binding entity comprises twoFab fragments each comprising a light chain and the C_(H1) and variableregions of a heavy chain, and the Fab fragments of the IL-23 bindingentity are linked to the C-termini of the heavy chains (Fc) of theIL-17A/F binding entity. This bispecific antibody format is referred toherein as biAbFabL (see FIG. 2). In another embodiment, each of thelight chain and the C_(H1) and variable regions of the heavy chaincomprising the Fab fragments of the IL-23 binding entity are linked tothe N-termini of the light chains and heavy chains, respectively, of theIL-17A/F binding entity. This bispecific antibody format is referred toherein as taFab (see FIG. 3).

In another aspect of the invention, the IL-23 binding entity comprisesan antibody, i.e., two pairs of immunoglobulin chains, each pair havingone light and one heavy chain, and the IL-17A/F binding entity comprisestwo Fab fragments each comprising a light chain and the C_(H1) andvariable regions of a heavy chain, and the Fab fragments of the IL-17A/Fbinding entity are linked to the C-termini of the heavy chains (Fc) ofthe IL-23 binding entity. This bispecific antibody format is referred toherein as biAbFabL (See FIG. 2). In another embodiment, each of thelight chain and the C_(H1) and variable regions of the heavy chaincomprising the Fab fragments of the IL-17A/F binding entity are linkedto the N-termini of the light chain and heavy chain, respectively, ofthe IL-23 binding entity. This bispecific antibody format is referred toherein as taFab (see FIG. 3).

In another aspect of the invention, the IL-23 binding entity comprises alight chain and an IL-23 heavy chain and the IL-17A/F binding entitycomprises a light chain and an IL-17A/F heavy chain. This bispecificantibody resembles a traditional antibody except that it comprises twodifferent heavy chains that associate through an electrostaticcomplementarity association in the C_(H3) regions. It utilizes a commonlight chain. This bispecific antibody format is referred to herein asHeterodimeric Fc (see FIG. 4).

In another embodiment, the present invention provides bispecificantibodies comprising a first binding entity comprising an antibody,i.e., two pairs of immunoglobulin chains, each pair having one lightchain and one heavy chain, and a second binding entity comprising an Fvunit, i.e., the variable domains from a heavy and a light chain, and inwhich the second binding entity comprising the Fv unit is positionedbetween the Fab region and the hinge of the first binding entity asshown in FIG. 5. The Fv unit is linked to the Fab region of the firstbinding entity by linker molecules. More specifically, the Fv unitcomprises a variable light domain which is linked to the light chainconstant region of the Fab fragment, and a variable heavy domain whichis linked to the C_(H1) region of the Fab fragment. This bispecificantibody format is referred to herein as VCVFc. The first binding entityand second binding entity of a VCVFc do not have to share a common lightchain, while the first binding entity and second binding entity of abiAbFabL do have to share a common light chain. In one aspect of thisembodiment of the invention, the first binding entity specifically bindsa lymphocyte antigen, cytokine, cytokine receptor, growth factor, growthfactor receptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23)or interleukin receptor and the second binding entity specifically bindsa lymphocyte antigen, cytokine, cytokine receptor, growth factor, growthfactor receptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23)or interleukin receptor. In another aspect of this embodiment of theinvention, the first binding entity is an IL-17A/F binding entity andthe second binding entity is an IL-23 binding entity. In another aspectof this embodiment of the invention, the first binding entity is anIL-23 binding entity and the second binding entity is an IL-17A/Fbinding entity.

In another embodiment, the present invention provides bispecificantibodies comprising a first binding entity comprising an antibody,i.e., two pairs of immunoglobulin chains, each pair having one lightchain and one heavy chain, and a second binding entity comprising asingle domain antibody. This bispecific antibody format is referred toherein as VCDFc. An illustration of a VCDFc bispecific antibody is shownin FIG. 6. The second binding entity comprising the single domainantibody is positioned between the Fab region, more specifically theC_(H1) region of the Fab fragment, and the hinge of the first bindingentity. The single domain antibody is linked to the C_(H1) region of theFab of the first binding entity by linker molecules (for example, butnot limited to, 10 mer G₄S, which is represented by the equation (G₄S)₂,or SSASTKGPS (SEQ ID NO:86)). In one aspect of this embodiment of theinvention, the first binding entity specifically binds a lymphocyteantigen, cytokine, cytokine receptor, growth factor, growth factorreceptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23) orinterleukin receptor and the second binding entity specifically binds alymphocyte antigen, cytokine, cytokine receptor, growth factor, growthfactor receptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23)or interleukin receptor. In one aspect of this embodiment of theinvention, the first binding entity is an IL-23 binding entity and thesecond binding entity is an IL-17A/F binding entity. In another aspectof this embodiment of the invention, the first binding entity is anIL-17A/F binding entity and the second binding entity is an IL-23binding entity.

The amino acid sequences of the binding entities are preferably basedupon the sequences of human and/or humanized monoclonal antibodiesagainst a lymphocyte antigen, cytokine, cytokine receptor, growthfactor, growth factor receptor, interleukin (e.g., IL-17A, IL-17F,IL-17A/F and IL-23) or interleukin receptor.

In one embodiment of the foregoing aspects of the invention, the lightchains of the IL-17A/F binding entity and the IL-23 binding entity ofthe bispecific antibody each comprise a variable domain comprising aCDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having theamino acid sequence of SEQ ID NO:23, and a CDR3 having the sequence ofSEQ ID NO:24. In another embodiment, the light chains of the IL-17A/Fbinding entity and the IL-23 binding entity each comprise a variabledomain comprising the amino acid sequence of SEQ ID NO:9. In anotherembodiment, the light chains of the IL-17A/F binding entity and theIL-23 binding entity each comprise a constant domain comprising theamino acid sequence of SEQ ID NO:10. In another embodiment, the lightchains of the IL-17A/F binding entity and the IL-23 binding entity eachcomprise a variable domain comprising the amino acid sequence of SEQ IDNO:9 and a constant domain comprising the amino acid sequence of SEQ IDNO:10.

In another embodiment of the foregoing aspects of the invention, theheavy chain of the IL-17A/F binding entity of the bispecific antibodycomprises a variable domain comprising a CDR1 having the amino acidsequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27. Inanother embodiment, the heavy chain of the IL-17A/F binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:13. In another embodiment, when the IL-17A/F binding entity comprisesan antibody, the heavy chain constant domain comprises the amino acidsequence of SEQ ID NO:8 or SEQ ID NO:11. In another embodiment, when theIL-17A/F binding entity comprises a Fab fragment, the C_(H1) region ofthe heavy chain comprises the amino acid sequence of SEQ ID NO:14 or SEQID NO:15.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F binding entity of the bispecific antibody comprises a heavychain variable domain having at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity with the amino acid sequenceof SEQ ID NO:13. Optionally, all of the substitutions, additions ordeletions are within the framework region of the heavy chain variabledomain. Optionally, the IL-17A/F binding entity of the bispecificantibody comprises a heavy chain variable domain having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:13, wherein the variabledomain comprises a CDR1 having the amino acid sequence of SEQ ID NO:25,a CDR2 having the amino acid sequence of SEQ ID NO:26, and a CDR3 havingthe amino acid sequence of SEQ ID NO:27. Optionally, the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO:13.Optionally, the three IL-17A/F heavy chain variable domain CDRs includea CDR1 region comprising an amino acid sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:25; a CDR2 region comprisingan amino acid sequence having at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity with the amino acid sequenceof SEQ ID NO:26; and a CDR3 region comprising an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:27.Optionally, the IL-17A/F heavy chain variable domain CDR1 has the aminoacid sequence of SEQ ID NO:25; the heavy chain variable domain CDR2 hasthe amino acid sequence of SEQ ID NO:26; and the heavy chain variabledomain CDR3 has the amino acid sequence of SEQ ID NO:27. The IL-17A/Fand/or IL-23p19 binding entity comprises a light chain variable domainhaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:9.Optionally, all of the substitutions, additions or deletions are withinthe framework region of the light chain variable domain. Optionally, theIL-17A/F and/or IL-23p19 binding entity of the bispecific antibodycomprises a light chain variable domain having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity with theamino acid sequence of SEQ ID NO:9, wherein the variable domaincomprises a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2having the amino acid sequence of SEQ ID NO:23, and a CDR3 having theamino acid sequence of SEQ ID NO:24. Optionally, the light chainvariable domain comprises the amino acid sequence of SEQ ID NO:9.Optionally, the three IL-17A/F and/or IL-23p19 light chain variabledomain CDRs include a CDR1 region comprising an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:22; aCDR2 region comprising an amino acid sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:23; and a CDR3 regioncomprising an amino acid sequence having at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% sequence identity with the aminoacid sequence of SEQ ID NO:24. Optionally, the IL-17A/F and/or IL-23p19light chain variable domain CDR1 has the amino acid sequence of SEQ IDNO:22; the IL-17A/F and/or IL-23p19 light chain variable domain CDR2 hasthe amino acid sequence of SEQ ID NO:23; and the IL-17A/F and/orIL-23p19 light chain variable domain CDR3 has the amino acid sequence ofSEQ ID NO:24. The IL-23p19 binding entity comprises a heavy chainvariable domain having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity with the amino acid sequenceof SEQ ID NO:7. Optionally, all of the substitutions, additions ordeletions are within the framework region of the IL-23p19 heavy chainvariable domain. Optionally, the IL-23p19 binding entity of thebispecific antibody comprises a heavy chain variable domain having atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity with the amino acid sequence of SEQ ID NO:7, whereinthe variable domain comprises a CDR1 having the amino acid sequence ofSEQ ID NO:19, a CDR2 having the amino acid sequence of SEQ ID NO:20, anda CDR3 having the amino acid sequence of SEQ ID NO:21. Optionally, theIL-23p19 heavy chain variable domain comprises the amino acid sequenceof SEQ ID NO:7. Optionally, the three IL-23p19 heavy chain variabledomain CDRs include a CDR1 region comprising an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:19; aCDR2 region comprising an amino acid sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:20; and a CDR3 regioncomprising an amino acid sequence having at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% sequence identity with the aminoacid sequence of SEQ ID NO:21. Optionally, the IL-23p19 heavy chainvariable domain CDR1 has the amino acid sequence of SEQ ID NO:19; theheavy chain variable domain CDR2 has the amino acid sequence of SEQ IDNO:20; and the heavy chain variable domain CDR3 has the amino acidsequence of SEQ ID NO:21.

In another embodiment of the foregoing aspects of the invention, theheavy chain of the IL-23 binding entity of the bispecific antibodycomprises a variable domain comprising a CDR1 having the amino acidsequence of SEQ ID NO:19, a CDR2 having the amino acid sequence of SEQID NO:20, and a CDR3 having the amino acid sequence of SEQ ID NO:21. Inanother embodiment, the heavy chain of the IL-23 binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:7. In another embodiment, when the IL-23 binding entity comprises anantibody, the heavy chain constant domain comprises the amino acidsequence of SEQ ID NO:8 or SEQ ID NO:11. In some embodiments, theC-terminal lysine of SEQ ID NO:8 has been cleaved, and so the heavychain constant domain comprises the amino acid sequence of residues1-326 of SEQ ID NO:8. In another embodiment, when the IL-23 bindingentity comprises a Fab fragment, the C_(H1) region of the heavy chaincomprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15.

In another embodiment of the foregoing aspects of the invention, whenthe IL-23 binding entity or IL-17A/F binding entity of the bispecificantibody is an Fv unit, the variable domain of the light chain comprisesa CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having theamino acid sequence of SEQ ID NO:23, and a CDR3 having the sequence ofSEQ ID NO:24. In another embodiment, the light chains of the IL-17A/Fbinding entity and the IL-23 binding entity each comprise a variabledomain comprising the amino acid sequence of SEQ ID NO:9.

In another embodiment of the foregoing aspects of the invention, whenthe IL-17A/F binding entity of the bispecific antibody is an Fv unit,the variable domain of the heavy chain comprises a CDR1 having the aminoacid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence ofSEQ ID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27.In another embodiment, the heavy chain of the IL-17A/F binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:13.

In another embodiment of the foregoing aspects of the invention, whenthe IL-23 binding entity of the bispecific antibody is an Fv unit, thevariable domain of the heavy chain comprises a CDR1 having the aminoacid sequence of SEQ ID NO:19, a CDR2 having the amino acid sequence ofSEQ ID NO:20, and a CDR3 having the amino acid sequence of SEQ ID NO:21.In another embodiment, the heavy chain of the IL-23 binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:7.

In another embodiment of the foregoing aspects of the invention, the Fabfragments of the IL-23 binding entity of the bispecific antibody arelinked to the C-termini of the heavy chains (Fc) of the IL-17A/F bindingentity, or the Fab fragments of the IL-17A/F binding entity are linked,for example, to the C-termini of the heavy chains (Fc) of the IL-23binding entity by a linker molecule (see, for example, FIG. 2). Inanother embodiment, each of the light chain and the C_(H1) and variableregions of the heavy chain comprising the Fab fragments of the IL-23binding entity are linked to the N-termini of the light chain and heavychain, respectively, of the IL-17A/F binding entity, or each of thelight chain and the C_(H1) and variable regions of the heavy chaincomprising the Fab fragments of the IL-17A/F binding entity are linkedto the N-termini of the light chain and heavy chain, respectively, ofthe IL-23 binding entity by a linker molecule (see, for example, FIG.3). In another embodiment of the VCVFc bispecific antibody, each of thelight chain variable region and the heavy chain variable region of theFv unit comprising the second binding entity are linked to each of thelight chain constant region and the C_(H1) region, respectively, of theFab fragment of the first binding entity by a linker molecule (see FIG.5). Suitable linker molecules are known in the art and include, forexample, short polypeptides. A suitable linker may include a shortpolypeptide, which contains glycine, which confers flexibility, andserine or threonine, which confer solubility. A suitable linker maycomprise Gly₄Ser₁ units. For example, the linker may be (Gly₄Ser₁)_(x),wherein x is 1, 2, or 3. Optionally, the linker polypeptide has theamino acid sequence of SEQ ID NO:12. In another embodiment of the VCVFcbispecific antibodies, the linker for the light chain has the amino acidsequence of SEQ ID NO:85 and the linker for the heavy chain has theamino acid sequence of SEQ ID NO:86.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises a pair of heavy chains each comprising theamino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:74, or SEQ ID NO:84 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:17. In a preferredembodiment, the bispecific antibody comprises a pair of heavy chainscomprising the amino acid sequence of SEQ ID NO:74 and a pair of lightchains each comprising the amino acid sequence of SEQ ID NO:17.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F antibody (or an antigen-binding fragment thereof) or theIL-17A/F binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds (a) an IL-17A homodimerwith a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹M, at least 1×10⁻¹⁰ M, at least 5×10⁻¹⁰ M, at least 8×10⁻¹⁰ M or atleast at least 1×10⁻¹¹ M; (b) an IL-17F homodimer with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰ M or at least 1×10⁻¹¹ M; and/or (c) an IL-17A/Fheterodimer with a binding affinity (K_(D1)) of at least 1×10⁻⁸ M, atleast 5×10⁻⁸ M, at least 1×10⁻⁹ M, at least 2×10⁻⁹ M, at least 3×10⁻⁹ M,at least 4×10⁻⁹ M, at least 5×10⁻⁹ M, at least 6×10⁻⁹ M, at least 7×10⁻⁹M, at least 9×10⁻⁹ M, at least 1×10⁻¹⁰ M or at least 5×10⁻¹⁰ M, whereinthe binding affinity is measured by surface plasmon resonance, such asBiacore.

In another embodiment of the foregoing aspects of the invention, anIL-23p19 antibody (or an antigen-binding fragment thereof) or theIL-23p19 binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds IL-23p19 with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰, at least 6×10⁻¹⁰, at least 7×10⁻¹⁰, at least 8×10⁻¹⁰or at least 9×10⁻¹⁰, at least 1×10⁻¹¹, wherein the binding affinity ismeasured by surface plasmon resonance, such as Biacore.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds (a) an IL-17A homodimerwith a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹M, at least 1×10⁻¹⁰ M, at least 5×10⁻¹⁰ M, at least 8×10⁻¹⁰ M or atleast at least 1×10⁻¹¹ M; (b) an IL-17F homodimer with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰ M or at least 1×10⁻¹¹ M; and/or (c) an IL-17A/Fheterodimer with a binding affinity (K_(D1)) of at least 1×10⁻⁸ M, atleast 5×10⁻⁸ M, at least 1×10⁻⁹ M, at least 2×10⁻⁹ M, at least 3×10⁻⁹ M,at least 4×10⁻⁹ M, at least 5×10⁻⁹ M, at least 6×10⁻⁹ M, at least 7×10⁻⁹M, at least 9×10⁻⁹ M, at least 1×10⁻¹⁰ M or at least 5×10⁻¹⁰ M; and theIL-23p19 binding entity of the bispecific antibody binds IL-23p19 with abinding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, atleast 1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least4×10⁻¹⁰ M, at least 5×10⁻¹⁰, at least 6×10⁻¹⁰, at least 7×10⁻¹⁰, atleast 8×10⁻¹⁰ or at least 9×10⁻¹⁰, at least 1×10⁻¹¹, wherein the bindingaffinity is measured by surface plasmon resonance, such as Biacore.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F antibody (or an antigen-binding fragment thereof) or theIL-17A/F binding entity of the bispecific antibody neutralizes orinhibits (a) IL-17A induction of G-CSF in primary human small airwayepithelial cells (SAEC) with an IC₅₀ of 0.5 pm or less; (b) IL-17Finduction G-CSF in primary human small airway epithelial cells (SAEC)with an IC₅₀ of 2.0 nM or less, 1.5 nM or less, 1.4 nM or less, 1.3 nMor less, 1.2 nM or less, 1.1 nM or less, or 1.0 nM or less; and/or (c)IL-17A/F induction G-CSF in primary human small airway epithelial cells(SAEC) with an IC₅₀ of 1.3 nM or less, 1.2 nM or less, 1.1 nM or less,1.0 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nMor less, or 0.5 nM or less.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F antibody (or an antigen-binding fragment thereof) or theIL-17A/F binding entity of the bispecific antibody neutralizes orinhibits (a) IL-17A induction of IL-6 in human primary fibroblast cells(HFFF) with an IC₅₀ of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less,0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nMor less, 0.02 nM or less, or 0.01 nM or less; (b) IL-17F induction ofIL-6 in human primary fibroblast cells (HFFF) with an IC₅₀ of 30 nM orless, 28 nM or less, 26 nM or less, 25 nM or less, 22 nM or less, 20 nMor less, 19 nM or less, 18 nM or less, 17 nM or less, 16 nM or less, 15nM or less, 14 nM or less, 13 nM or less, 12 nM or less, 11 nM or less,or 10 nM or less; and/or (c) IL-17A/F induction of IL-6 in human primaryfibroblast cells (HFFF) with an IC₅₀ of 30 nM or less, 28 nM or less, 26nM or less, 22 nM or less, 20 nM or less, 18 nM or less, 17 nM or less,16 nM or less, 15 nM or less, 14 nM or less, 13 nM or less, 12 nM orless, 11 nM or less, 10 nM or less, 9.5 nM or less, 9.4 nM or less, 9.3nM or less, 9.2 nM or less, 9.1 nM or less, or 9.0 nM or less.

In another embodiment of the foregoing aspects of the invention, anIL-23p19 antibody (or an antigen-binding fragment thereof) or theIL-23p19 binding entity of the bispecific antibody neutralizes orinhibits (a) IL-23 induced IL-17A and IL-17F production in murinesplenocytes with an IC₅₀ of 0.5 nM or less, 0.4 nM or less, 0.3 nM orless, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less,0.07 nM or less, or 0.06 nM or less.

In another embodiment of the foregoing aspects of the invention, anIL-23p19 antibody (or an antigen-binding fragment thereof) or theIL-23p19 binding entity of the bispecific antibody neutralizes orinhibits IL-23 induced STAT3 phosphorylation in activated primary humanT cells with an IC₅₀ of 0.1 nM or less, 0.2 nM or less, 0.3 nM or less,0.4 nM or less, 0.5 nM or less, 0.8 nM or less, 0.9 nM or less, 0.01 nMor less, 0.02 nM or less, 0.03 nM or less, 0.04 nM or less, or 0.05 nMor less.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds (a) an IL-17A homodimerwith a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹M, at least 1×10⁻¹⁰ M, at least 5×10⁻¹⁰ M, at least 8×10⁻¹⁰ M or atleast at least 1×10⁻¹¹ M; (b) an IL-17F homodimer with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰ M or at least 1×10⁻¹¹ M; and/or (c) an IL-17A/Fheterodimer with a binding affinity (K_(D1)) of at least 1×10⁻⁸ M, atleast 5×10⁻⁸ M, at least 1×10⁻⁹ M, at least 2×10⁻⁹ M, at least 3×10⁻⁹ M,at least 4×10⁻⁹ M, at least 5×10⁻⁹ M, at least 6×10⁻⁹ M, at least 7×10⁻⁹M, at least 9×10⁻⁹ M, at least 1×10⁻¹⁰ M or at least 5×10⁻¹⁰ M.Optionally, the IL-23p19 binding entity of the bispecific antibody bindsIL-23p19 with a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least5×10⁻⁹ M, at least 1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, atleast 4×10⁻¹⁰ M, at least 5×10⁻¹⁰, at least 6×10⁻¹⁰, at least 7×10⁻¹⁰,at least 8×10⁻¹⁰ or at least 9×10⁻¹⁰, at least 1×10⁻¹¹, wherein thebinding affinity is measured by surface plasmon resonance, such asBiacore. Optionally, the IL-17A/F binding entity of the bispecificantibody neutralizes or inhibits (a) IL-17A induction of G-CSF inprimary human small airway epithelial cells (SAEC) with an IC₅₀ of 0.5pm or less; (b) IL-17F induction G-CSF in primary human small airwayepithelial cells (SAEC) with an IC₅₀ of 2.0 nM or less, 1.5 nM or less,1.4 nM or less, 1.3 nM or less, 1.2 nM or less, 1.1 nM or less, or 1.0nM or less; and/or (c) IL-17A/F induction G-CSF in primary human smallairway epithelial cells (SAEC) with an IC₅₀ of 1.3 nM or less, 1.2 nM orless, 1.1 nM or less, 1.0 nM or less, 0.9 nM or less, 0.8 nM or less,0.7 nM or less, 0.6 nM or less, or 0.5 nM or less. Optionally, theIL-17A/F binding entity of the bispecific antibody neutralizes orinhibits (a) IL-17A induction of IL-6 in human primary fibroblast cells(HFFF) with an IC₅₀ of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less,0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nMor less, 0.02 nM or less, or 0.01 nM or less; (b) IL-17F induction ofIL-6 in human primary fibroblast cells (HFFF) with an IC₅₀ of 30 nM orless, 28 nM or less, 26 nM or less, 25 nM or less, 22 nM or less, 20 nMor less, 19 nM or less, 18 nM or less, 17 nM or less, 16 nM or less, 15nM or less, 14 nM or less, 13 nM or less, 12 nM or less, 11 nM or less,or 10 nM or less; and/or (c) IL-17A/F induction of IL-6 in human primaryfibroblast cells (HFFF) with an IC₅₀ of 30 nM or less, 28 nM or less, 26nM or less, 22 nM or less, 20 nM or less, 18 nM or less, 17 nM or less,16 nM or less, 15 nM or less, 14 nM or less, 13 nM or less, 12 nM orless, 11 nM or less, 10 nM or less, 9.5 nM or less, 9.4 nM or less, 9.3nM or less, 9.2 nM or less, 9.1 nM or less, or 9.0 nM or less.Optionally, the IL-23p19 binding entity of the bispecific antibodyneutralizes or inhibits IL-23 induced STAT3 phosphorylation in activatedprimary human T cells with an IC₅₀ of 0.1 nM or less, 0.2 nM or less,0.3 nM or less, 0.4 nM or less, 0.5 nM or less, 0.8 nM or less, 0.9 nMor less, 0.01 nM or less, 0.02 nM or less, 0.03 nM or less, 0.04 nM orless, or 0.05 nM or less.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises a pair of heavy chains comprising theamino acid sequence of SEQ ID NO:28 and a pair of light chainscomprising the amino acid sequence of SEQ ID NO:17 is referred to hereinas “biAb1”. A bispecific antibody comprising a pair of heavy chains eachcomprising the amino acid sequence of SEQ ID NO:18 and a pair of lightchains each comprising the amino acid sequence of SEQ ID NO:17 isreferred to herein as “biAb2”. A bispecific antibody comprising a pairof heavy chains each comprising the amino acid sequence of SEQ ID NO:74and a pair of light chains each comprising the amino acid sequence ofSEQ ID NO:17 is referred to herein as “biAb3”.

A bispecific antibody comprising a pair of heavy chains comprising theamino acid sequence of SEQ ID NO:29 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:17 is referred to hereinas “biAb4”.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises a pair of heavy chains each comprising theamino acid sequence of SEQ ID NO:77 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:79 and is referred toherein as “taFab1”.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises an IL-23 heavy chain comprising the aminoacid sequence of SEQ ID NO:63, an IL-17A/F heavy chain comprising theamino acid sequence of SEQ ID NO:65, and a pair of light chains eachcomprising the sequence of SEQ ID NO:17, and is referred to herein as“hetero1”. In another embodiment, the bispecific antibody comprises anIL-23 heavy chain comprising the amino acid sequence of SEQ ID NO:61, anIL-17A/F heavy chain comprising the amino acid sequence of SEQ ID NO:81,and a pair of light chains each comprising the sequence of SEQ ID NO:17,and is referred to herein as “hetero2”.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody in the VCVFc format, see FIG. 5, has a pair of heavychains each comprising the amino acid sequence of SEQ ID NO:88 and apair of light chains each comprising the amino acid sequence of SEQ IDNO:90, or a pair of heavy chains each comprising the amino acid sequenceof SEQ ID NO:92 and a pair of light chains each comprising the aminoacid sequence of SEQ ID NO:94, or a pair of heavy chains each comprisingthe amino acid sequence of SEQ ID NO:96 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:90, or a pair of heavychains each comprising the amino acid sequence of SEQ ID NO:98 and apair of light chains each comprising the amino acid sequence of SEQ IDNO:94, or a pair of heavy chains each comprising the amino acid sequenceof SEQ ID NO:100 and a pair of light chains each comprising the aminoacid sequence of SEQ ID NO:102, or a pair of heavy chains eachcomprising the amino acid sequence of SEQ ID NO:104 and a pair of lightchains each comprising the amino acid sequence of SEQ ID NO:106, or apair of heavy chains each comprising the amino acid sequence of SEQ IDNO:112 and a pair of light chains each comprising the amino acidsequence of SEQ ID NO:114, or a pair of heavy chains each comprising theamino acid sequence of SEQ ID NO:116 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:118.

In another embodiment of the foregoing aspects of the invention, anisolated monoclonal antibody or antigen-binding fragment thereof thatspecifically binds to IL-17A (SEQ ID NO:2) and IL-17F (SEQ ID NO:4)comprising a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises the amino acidresidues of SEQ ID NO:13 and a light chain variable domain comprises theamino acid residues of SEQ ID NO:9. Optionally, the monoclonal antibodycomprises a human constant region, e.g., IgG1, IgG2, IgG3 or IgG4. TheIgG4 human constant region may have a Serine to Proline mutation atposition 241 according to Kabat. Optionally, the heavy chain comprisesthe amino acid residues of SEQ ID NOs:16, 18, 28, 29 or 74. Optionally,the light chain comprises the amino acid residues of SEQ ID NO:17.Optionally, the heavy chain comprises the amino acid residues of SEQ IDNOs:16, 18, 28, 29 or 74, and the light chain comprises the amino acidresidues of SEQ ID NO:17. Optionally, a bispecific antibody comprisesthe monoclonal antibody.

In another embodiment of the foregoing aspects of the invention, anisolated monoclonal antibody or antigen-binding fragment thereof thatspecifically binds to IL-23p19 (SEQ ID NO:6) comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises the amino acid residues of SEQ ID NO:7,and wherein the light chain variable domain comprises the amino acidresidues of SEQ ID NO:9. Optionally, the monoclonal antibody comprises ahuman constant region, e.g., IgG1, IgG2, IgG3 or IgG4. Optionally, theIgG4 human constant region has a Serine to Proline mutation at position241 according to Kabat. Optionally, the heavy chain comprises the aminoacid residues of SEQ ID NOs:16, 18, 28, 29 or 74. Optionally, the lightchain comprises the amino acid residues of SEQ ID NO:17. Optionally, theheavy chain comprises the amino acid residues of SEQ ID NOs:16, 18, 28,29 or 74, and the light chain comprises the amino acid residues of SEQID NO:17. Optionally, a bispecific antibody comprises the monoclonalantibody.

In another embodiment of the foregoing aspects of the invention, theantibody, bispecific antibody, or antigen-binding fragment thereof,specifically binds IL-23p19, wherein the antibody or antigen-bindingfragment binds a discontinuous epitope on IL-23p19 comprising a firstepitope and a second epitope, wherein the first epitope consists of atleast one amino acid of amino acid residues 33-59 of SEQ ID NO:6 and thesecond epitope consists of at least one amino acid of amino acidresidues 89-125 of SEQ ID NO:6. Optionally, the antibody, bispecificantibody, or antigen-binding fragment thereof binds to at least aminoacid residue 54 of SEQ ID NO:6 of the first epitope. Optionally, theantibody, bispecific antibody, or antigen-binding fragment thereof bindsto at least amino acid residue 55 of SEQ ID NO:6 of the first epitope.Optionally, the antibody, bispecific antibody, or antigen-bindingfragment thereof binds to at least amino acid residues 54 and 55 of SEQID NO:6 of the first epitope. Optionally, the antibody, bispecificantibody, or antigen-binding fragment thereof binds to at least aminoacid residue 116 of SEQ ID NO:6 of the second epitope. Optionally, theantibody, bispecific antibody, or antigen-binding fragment thereof bindsto at least amino acid residues 54 and 55 of SEQ ID NO:6 of the firstepitope, and to least amino acid residue 116 of SEQ ID NO:6 of thesecond epitope.

In another embodiment of the foregoing aspects of the invention, theantibody, bispecific antibody, or antigen-binding fragment thereofspecifically binds IL-23p19, wherein the antibody or antigen-bindingfragment binds a discontinuous epitope on IL-23p19 comprising a firstepitope and a second epitope, wherein the antibody or antigen-bindingfragment binds to at least amino acid residues 54 and 55 of SEQ ID NO:6of the first epitope, and to least amino acid residue 116 of SEQ ID NO:6of the second epitope.

The bispecific antibodies of the invention may be used alone or asimmunoconjugates with a cytotoxic agent. In some embodiments, the agentis a chemotherapeutic agent. In some embodiments, the agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, abrin, modified Pseudomonas enterotoxin A, Pseudomonasexotoxin, calicheamicin, adriamycin, 5-fluorouracil, diphtheria toxin,and the like. Methods of conjugation of antibodies to such agents areknown in the literature, and include direct and indirect conjugation.

Suitable detectable molecules may be directly or indirectly attached tothe antibodies of the present invention. Suitable detectable moleculesinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like. For indirect attachment of a detectable or cytotoxic molecule,the detectable or cytotoxic molecule can be conjugated with a member ofa complementary/anticomplementary pair, where the other member is boundto the binding polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

The bispecific antibodies, antibodies and antigen-binding fragments ofthe invention also include derivatives that are modified, e.g., by thecovalent attachment of any type of molecule to the antibody such thatcovalent attachment does not prevent the antibody from binding to itsepitope. Examples of suitable derivatives include, but are not limitedto fucosylated antibodies, glycosylated antibodies, acetylatedantibodies, pegylated antibodies, phosphorylated antibodies, andamidated antibodies. The antibodies and derivatives thereof of theinvention may themselves by derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The bispecific antibodies, antibodies and antigen-binding fragments ofthe invention include variants having single or multiple amino acidsubstitutions, deletions, additions, or replacements that retain thebiological properties (e.g., block the binding of IL-17A or IL-17Fand/or IL-23 to their respective receptors, inhibit the biologicalactivity of IL-17A or IL-17F and IL-23) of the antibodies of theinvention. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include, inter alia: (a) variants in which one ormore amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art.

The invention also includes isolated nucleic acids encoding thebispecific antibodies of the invention, which includes, for instance,the light chain, light chain variable region, light chain constantregion, heavy chain, heavy chain variable region, heavy chain constantregion, linkers, and any and all components and combinations thereof ofthe bispecific antibodies disclosed herein. Nucleic acids of theinvention include nucleic acids having at least 80%, more preferably atleast about 90%, more preferably at least about 95%, and most preferablyat least about 98% homology to nucleic acids of the invention. The terms“percent similarity”, “percent identity” and “percent homology” whenreferring to a particular sequence are used as set forth in theUniversity of Wisconsin GCG® software program. Nucleic acids of theinvention also include complementary nucleic acids. In some instances,the sequences will be fully complementary (no mismatches) when aligned.In other instances, there may be up to about a 20% mismatch in thesequences. In some embodiments of the invention are provided nucleicacids encoding both a heavy chain and a light chain of an antibody ofthe invention.

Nucleic acids of the invention can be cloned into a vector, such as aplasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) orvirus, into which another genetic sequence or element (either DNA orRNA) may be inserted so as to bring about the replication of theattached sequence or element. In some embodiments, the expression vectorcontains a constitutively active promoter segment (such as but notlimited to CMV, SV40, Elongation Factor or LTR sequences) or aninducible promoter sequence such as the steroid inducible pIND vector(Invitrogen), where the expression of the nucleic acid can be regulated.Expression vectors of the invention may further comprise regulatorysequences, for example, an internal ribosomal entry site. The expressionvector can be introduced into a cell by transfection, for example.

Thus in another embodiment, the present invention provides an expressionvector comprising the following operably linked elements; atranscription promoter; a nucleic acid molecule encoding the heavy chainof a bispecific antibody of the invention; and a transcriptionterminator. In another embodiment, the present invention provides anexpression vector comprising the following operably linked elements; atranscription promoter; a nucleic acid molecule encoding the light chainof a bispecific antibody of the invention; and a transcriptionterminator. Recombinant host cells comprising such vectors andexpressing the heavy and light chains are also provided.

In another embodiment, the present invention provides an expressionvector comprising the following operably linked elements; atranscription promoter; a first nucleic acid molecule encoding the heavychain of a bispecific antibody, antibody or antigen-binding fragment ofthe invention; a second nucleic acid molecule encoding the light chainof a bispecific antibody, antibody or antigen-binding fragment of theinvention; and a transcription terminator. In another embodiment, thepresent invention provides an expression vector comprising the followingoperably linked elements; a first transcription promoter; a firstnucleic acid molecule encoding the heavy chain of a bispecific antibody,antibody or antigen-binding fragment of the invention; a firsttranscription terminator; a second transcription promoter a secondnucleic acid molecule encoding the light chain of a bispecific antibody,antibody or antigen-binding fragment of the invention; and a secondtranscription terminator. Recombinant host cells comprising such vectorsand expressing the heavy and light chains are also provided.

Antibody-producing cells containing a nucleic acid encoding the heavychain and a nucleic acid encoding the light chain of the bispecificantibodies, antibodies or antigen-binding fragments of the invention canbe used to produce the bispecific antibodies, antibodies orantigen-binding fragments in accordance with techniques known in theart. The present invention, in one embodiment, provides a method ofproducing a bispecific antibody, antibody or antigen-binding fragment ofthe invention comprising culturing a recombinant host cell expressingthe heavy and light chains and isolating the bispecific antibody,antibody or antigen-binding fragment produced by the cell.

The recombinant host cell may be a prokaryotic cell, for example a E.coli cell, or a eukaryotic cell, for example a mammalian cell or a yeastcell. Yeast cells include Saccharomyces cerevisiae, Schizosaccharomycespombe, and Pichia pastoris cells. Mammalian cells include VERO, HeLa,Chinese hamster Ovary (CHO), W138, baby hamster kidney (BHK), COS-7,MDCK, human embryonic kidney line 293, normal dog kidney cell lines,normal cat kidney cell lines, monkey kidney cells, African green monkeykidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells,fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTK31cells, mouse sertoli cells, human cervical carcinoma cells, buffalo ratliver cells, human lung cells, human liver cells, mouse mammary tumorcells, TRI cells, MRC 5 cells, and FS4 cells. Antibody-producing cellsof the invention also include any insect expression cell line known,such as for example, Spodoptera frugiperda cells. In a preferredembodiment, the cells are mammalian cells. In another preferredembodiment, the mammalian cells are CHO cells.

The antibody-producing cells preferably are substantially free ofIL-17A, IL-17F and IL-23 binding competitors. In preferred embodiments,the antibody-producing cells comprise less than about 10%, preferablyless than about 5%, more preferably less than about 1%, more preferablyless than about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight IL-17A, IL-17F, or IL-23 binding competitors. Insome embodiments, the antibodies produced by the antibody-producingcells are substantially free of IL-17A, IL-17F, and IL-23 competitors.In preferred embodiments, antibodies produced by the antibody-producingcells comprise less than about 10%, preferably less than about 5%, morepreferably less than about 1%, more preferably less than about 0.5%,more preferably less than about 0.1%, and most preferably 0% by weightboth IL-17 and IL-23 binding competitors.

Methods of antibody purification are known in the art. In someembodiments of the invention, methods for antibody purification includefiltration, affinity column chromatography, cation exchangechromatography, anion exchange chromatography, and concentration. Thefiltration step preferably comprises ultrafiltration, and morepreferably ultrafiltration and diafiltration. Filtration is preferablyperformed at least about 5-50 times, more preferably 10 to 30 times, andmost preferably 14 to 27 times. Affinity column chromatography, may beperformed using, for example, PROSEP® Affinity Chromatography(Millipore, Billerica, Mass.). In a preferred embodiment, the affinitychromatography step comprises PROSEP®-vA column chromatography. Eluatemay be washed in a solvent detergent. Cation exchange chromatography mayinclude, for example, SP-Sepharose Cation Exchange Chromatography. Anionexchange chromatography may include, for example but not limited to,Q-Sepharose Fast Flow Anion Exchange. The anion exchange step ispreferably non-binding, thereby allowing removal of contaminantsincluding DNA and BSA. The antibody product is preferably nanofiltered,for example, using a Pall DV 20 Nanofilter. The antibody product may beconcentrated, for example, using ultrafiltration and diafiltration. Themethod may further comprise a step of size exclusion chromatography toremove aggregates.

The bispecific antibodies, antibodies or antigen-binding fragments mayalso be produced by other methods known in the art, for example bychemical coupling of antibodies and antibody fragments.

The bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention are useful, for example, for the inhibition ofproinflammatory cytokines, such as IL-17A, IL-17F and IL-23/p19. Theantibodies can be used to reduce, limit, neutralize, or block theproinflammatory effects of the IL-17A homodimer, the IL-17F homodimer,and/or the IL-17A/F heterodimer. Likewise, the antibodies can be used toreduce, limit, neutralize, or block the pro-cancerous effects of theIL-17A homodimer, the IL-17F homodimer, or the IL-17A/F heterodimer. Insuch cases, the anti-IL-23p19 portion of the antibody is used to reduce,limit, neutralize, or block production of new T cells that would produceIL-17A and/or IL-17F, including homodimers and heterodimers. Thebispecific antibodies, antibodies or antigen-binding fragments describedherein can be used to treat inflammatory disorders and autoimmunediseases, such as multiple sclerosis, cystic fibrosis, inflammatorybowel disease, psoriasis, systemic sclerosis, systemic lupuserythematosus, lupus nephritis, IgA nephropathy, diabetic kidneydisease, minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis (FSGS), nephrogenic systemic fibrosis (NSF),nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis,eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popularmucinosis), scleroderma, lichen sclerosusetatrophicus, POEMs syndrome(Crow-Fukase syndrome, Takatsuki disease or PEP syndrome), nephroticsyndrome, graft-versus-host-disease (GVHD), graft-versus-host-disease(GVHD) (from a transplant, such as blood, bone marrow, kidney, pancreas,liver, orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis (AAV), giant cell arteritis andmultiple-myeloma-induced lytic bone disease. The bispecific antibodies,antibodies or antigen-binding fragments described herein can also beused to treat cancer, including angiogenesis.

The bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention inhibit the activity of IL-17A and/or IL-17F andIL-23 (via the p19 subunit), and thus, inhibit the production,maintenance, and activity of new and existing IL-17A and IL-17F andIL-17-producing T cells (Th17). The invention further concerns the useof the bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention in the treatment of inflammatory diseasescharacterized by the presence of elevated levels of IL-17A, IL-17F,and/or IL-23, and in the treatment of cancers characterized by thepresence of elevated levels of IL-17A, IL-17F, and/or IL-23.

The bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention may block, inhibit, reduce, antagonize orneutralize the activity of IL-17A, IL-17F, (including both homodimersand the heterodimer), and IL-23/p19 thus are advantageous over therapiesthat target only one or two of these three cytokines.

The antibodies, e.g., bispecific antibodies, of the invention are thususeful to:

(1) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A or IL-17F and IL-23 in the treatment of cancer, acuteinflammation, and chronic inflammatory diseases such as inflammatorybowel disease (IBD), Crohn's disease, ulcerative colitis, irritablebowel syndrome (IBS), cystic fibrosis, chronic colitis, Sjögren'ssyndrome, splenomegaly, inflammation in chronic kidney disease (CKD),psoriasis, psoriatic arthritis, rheumatoid arthritis, and other diseasesassociated with the induction of acute-phase response.

(2) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A or IL-17F or IL-23 in the treatment of autoimmune diseases suchas insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS),systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, Sjögren's syndrome, IBS and IBD to prevent or inhibitsignaling in immune cells (e.g., lymphocytes, monocytes, leukocytes) viatheir receptors (e.g., IL-23Rα, IL-12Rβ1, IL-17RA and IL-17RC).Blocking, inhibiting, reducing, or antagonizing signaling via IL-23Rα,IL-12Rβ1, IL-17RA and IL-17RC, using the antibodies of the presentinvention, also benefits diseases of the pancreas, kidney, pituitary andneuronal cells and may be used to treat IDDM, non-insulin dependentdiabetes mellitus (NIDDM), pancreatitis, and pancreatic carcinoma.

For example, the bispecific antibodies, antibodies or antigen-bindingfragments of the present invention are useful in therapeutic treatmentof inflammatory diseases, particularly as antagonists to IL-17A, IL-17F,and IL-23/p19, in the treatment of inflammatory diseases such asmultiple sclerosis (MS), inflammatory bowel disease (IBD), and cancer.These antagonists are capable of binding, blocking, inhibiting,reducing, antagonizing or neutralizing IL-17A, IL-17F, their homodimersand heterodimers, and IL-23 (via p19) (either individually or together)in the treatment of atopic and contact dermatitis, systemic sclerosis,systemic lupus erythematosus (SLE), antineutrophil cytoplasmicantibodies (ANCA)-associated vasculitis (AAV), giant cell arteritis,multiple sclerosis (MS), colitis, endotoxemia, arthritis, rheumatoidarthritis (RA), Sjögren's syndrome, psoriatic arthritis, adultrespiratory disease (ARD), septic shock, multiple organ failure,inflammatory lung injury such as idiopathic pulmonary fibrosis, asthma,chronic obstructive pulmonary disease (COPD), airwayhyper-responsiveness, chronic bronchitis, allergic asthma, psoriasis,eczema, IBS and inflammatory bowel disease (IBD) such as ulcerativecolitis and Crohn's disease, Helicobacter pylori infection, lupusnephritis, IgA nephropathy, diabetic kidney disease, minimal changedisease (lipoid nephrosis), focal segmental glomerulosclerosis (FSGS),nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy,fibrosing cholestatic hepatitis, eosinophilic fasciitis (Shulman'ssyndrome), scleromyxedema (popular mucinosis), scleroderma, lichensclerosusetatrophicus, POEMs syndrome (Crow-Fukase syndrome, Takatsukidisease or PEP syndrome), nephrotic syndrome, transplant rejection,graft-versus-host-disease (GVHD), graft-versus-host-disease (GVHD) (froma transplant, such as blood, bone marrow, kidney, pancreas, liver,orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), intraabdominal adhesions and/or abscessesas results of peritoneal inflammation (e.g., from infection, injury,etc.), nephrotic syndrome, cystic fibrosis (Tan, H.-L. et al., AmericanJournal of Respiratory and Critical Care Medicine, 184(2):252-258(2011)), lytic bone disease (e.g., multiple-myeloma-induced lytic bonedisease) (Sotomayor, E. M., Blood, 116(18):3380-3382 (2010)), organallograft rejection, streptococcal cell wall (SCW)-induced arthritis,osteoarthritis, gingivitis/periodontitis, herpetic stromal keratitis,restenosis, Kawasaki disease, age-related macular degeneration (AMD;e.g., wet form of AMD and dry form of AMD) (Wei, L. et al., CellReports, 2:1151-1158 (Nov. 29, 2012), immune mediated renal diseases,liver fibrosis (Meng, F. et al., Gastroenterology, 143:765-776 (2012),pulmonary fibrosis (Meng, F. et al., Gastroenterology, 143:765-776(2012), hepatobiliary diseases, myocarditis (Ding, H.-S., Mol. Biol.Rep., 39(7):7473-7478 (Feb. 14, 2012); Valente, A. J. et al., CellularSignalling, 24:560-568 (2012)), cardiac fibrosis (Valente, A. J. et al.,Cellular Signalling, 24:560-568 (2012)), adverse myocardial remodeling(Valente, A. J. et al., Cellular Signalling, 24:560-568 (2012)),atherosclerosis (Ding, H.-S., Mol. Biol. Rep., 39(7):7473-7478 (Feb. 14,2012), cardiac ischemia/reperfusion injury (Ding, H.-S., Mol. Biol.Rep., 39(7):7473-7478 (Feb. 14, 2012), heart failure (Ding, H.-S., Mol.Biol. Rep., 39(7):7473-7478 (Feb. 14, 2012) and cancers/neoplasticdiseases that are characterized by IL-17 and/or IL-23 expression,including but not limited to prostate, renal, colon, ovarian andcervical cancer, and leukemias (Tartour et al., Cancer Res., 59:3698(1999); Kato et al., Biochem. Biophys. Res. Commun., 282:735 (2001);Steiner et al., Prostate, 56:171 (2003); Langowksi et al., Nature, May10 [Epub ahead of print], (2006)).

For example, the bispecific antibodies, antibodies or antigen-bindingfragments of the present invention are useful, e.g., antagonists toIL-17A, IL-17F, and IL-23/p19, in therapeutic treatment of inflammatorydiseases, particularly in the treatment of Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Degos' disease,dermatomyositis-juvenile, discoid lupus (e.g., childhood discoid lupuserythematosus, generalized discoid lupus erythematosus and localizeddiscoid lupus erythematosus), chilblain lupus erythematosus, lupuserythematosus-lichen planus overlap syndrome, lupus erythematosuspanniculitis, tumid lupus erythematosus, verrucous lupus erythematosuscutaneous, systemic lupus erythematosus, subacute cutaneous lupuserythematosus, acute cutaneous lupus erythematosus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, rheumatoid arthritis (RA),Meniere's disease, mixed connective tissue disease, multiple sclerosis(MS), myasthenia gravis, pernacious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, eczema, psoriasis, psoriatic arthritis, Raynaud'sphenomena, Reiter's syndrome, adult respiratory disease (ARD), rheumaticfever, arthritis, sarcoidosis, scleroderma (e.g., progressive systemicsclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren'ssyndrome, stiff-man syndrome, systemic lupus erythematosus (SLE),antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis(AAV), giant cell arteritis, Takayasu arteritis, temporalarteritis/giant cell arteritis, endotoxia, sepsis or septic shock, toxicshock syndrome, multiple organ failure, inflammatory lung injury such asidiopathic pulmonary fibrosis, colitis, inflammatory bowel disease (IBD)such as ulcerative colitis and Crohn's disease, irritable bowel syndrome(IBS), uveitis, vitiligo, Wegener's granulomatosis, Alzheimer's disease,atopic allergy, allergy, asthma, bronchial asthma, chronic obstructivepulmonary disease (COPD), airway hyper-responsiveness, allergic asthma,glomerulonephritis, hemolytic anemias, Helicobacter pylori infection,intraabdominal adhesions and/or abscesses as results of peritonealinflammation (e.g., from infection, injury, etc.), nephrotic syndrome,idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, organallograft rejection, lupus nephritis, IgA nephropathy, diabetic kidneydisease, minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis (FSGS), nephrogenic systemic fibrosis (NSF),nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis,eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popularmucinosis), scleroderma, lichen sclerosusetatrophicus, POEMs syndrome(Crow-Fukase syndrome, Takatsuki disease or PEP syndrome), nephroticsyndrome, graft-versus-host-disease (GVHD), graft-versus-host-disease(GVHD) (from a transplant, such as blood, bone marrow, kidney, pancreas,liver, orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), lytic bone disease (e.g., multiplemyeloma-induced lytice bone disease), cystic fibrosis, age-relatedmascular degeneration (AMD; e.g., wet AMD and dry AMD), liver fibrosis,pulmonary fibrosis, atherosclerosis, cardiac ischemia/reperfusioninjury, heart failure, myocarditis, cardiac fibrosis, adverse myocardialremodeling, diabetic retinopathy and ventilator induced lung injury.

Accordingly, in one embodiment, the present invention provides a methodof inhibiting one or more of proinflammatory cytokines, e.g., IL-17A,IL-17F and IL-23, in a mammal in need of such treatment comprisingadministering a therapeutically effective amount of a bispecificantibody, antibody or antigen-binding fragment to a mammal in need ofsuch treatment. In a preferred embodiment, the mammal is a human. Themethod may be used to treat a disorder characterized by elevatedexpression of IL-17A, IL-17F, or IL-23. The bispecific antibody,antibody or antigen-binding fragment maybe administered with anotherpharmaceutical agent, either in the same formulation or separately.

In another embodiment, the present invention provides a method oftreating an immune related disorder in a mammal in need thereof,comprising administering to the mammal a therapeutically effectiveamount of an IL-17A/F polypeptide, an agonist thereof, or an antagonist(such as an IL-17A/F binding entity which includes an IL-17A/Fcross-reactive antibody) thereto. In a preferred aspect, the immunerelated disorder is selected form the group consisting of: systemiclupus erythematosis (SLE), rheumatoid arthritis (RA), osteoarthritis,juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis,idiopathic inflammatory myopathies, Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renaldisease, demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis (MS), idiopathic demyelinatingpolyneuropathy or Guillain-Barre syndrome, and chronic inflammatorydemyelinating polyneuropathy, hepatobiliary diseases such as infectious,autoimmune chronic active hepatitis, primary biliary cirrhosis,granulomatous hepatitis, and sclerosing cholangitis, inflammatory boweldisease (IBD), Crohn's disease, ulcerative colitis, gluten-sensitiveenteropathy, and Whipple's disease, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis (AAV), giant cell arteritis, psoriasis,psoriatic arthritis, allergic diseases such as asthma, allergicrhinitis, atopic dermatitis, food hypersensitivity and urticaria,immunologic diseases of the lung such as eosinophilic pneumonia,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, lupusnephritis, IgA nephropathy, diabetic kidney disease, minimal changedisease (lipoid nephrosis), focal segmental glomerulosclerosis (FSGS),nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy,fibrosing cholestatic hepatitis, eosinophilic fasciitis (Shulman'ssyndrome), scleromyxedema (popular mucinosis), scleroderma, lichensclerosusetatrophicus, POEMs syndrome (Crow-Fukase syndrome, Takatsukidisease or PEP syndrome), nephrotic syndrome, graft-versus-host-disease(GVHD), graft-versus-host-disease (GVHD) (from a transplant, such asblood, bone marrow, kidney, pancreas, liver, orthotopic liver, lung,heart, intestine, small intestine, large intestine, thymus, allogeneicstem cell, reduced-intensity allogeneic, bone, tendon, cornea, skin,heart valves, veins, arteries, blood vessels, stomach and testis), lyticbone disease (e.g., multiple myeloma-induced lytice bone disease),cystic fibrosis, age-related mascular degeneration (AMD; e.g., wet AMDand dry AMD), liver fibrosis, pulmonary fibrosis, atherosclerosis,cardiac ischemia/reperfusion injury, heart failure, myocarditis, cardiacfibrosis, adverse myocardial remodeling, transplantation associateddiseases including graft rejection and graft-versus-host-disease.

In another embodiment, the present invention provides a method forinhibiting inflammation in a mammal in need of such treatment comprisingadministering a therapeutically effective amount of a bispecificantibody, antibody or antigen-binding fragment of the invention to amammal in need of such treatment. In a preferred embodiment, the mammalis a human. The inflammation may be associated with a disease selectedfrom the group consisting of multiple sclerosis (MS), chronicinflammation, Sjögren's syndrome, autoimmune diabetes, rheumatoidarthritis (RA) and other arthritic conditions, asthma, systemicsclerosis, atopic dermatitis, antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis (AAV), giant cell arteritis, systemic lupuserythematosus (SLE), Degos' disease, dermatomyositis-juvenile, discoidlupus (e.g., childhood discoid lupus erythematosus, generalized discoidlupus erythematosus and localized discoid lupus erythematosus),chilblain lupus erythematosus, lupus erythematosus-lichen planus overlapsyndrome, lupus erythematosus panniculitis, tumid lupus erythematosus,verrucous lupus erythematosus cutaneous, systemic lupus erythematosus,subacute cutaneous lupus erythematosus, acute cutaneous lupuserythematosus, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, Graves' disease, lytic bone disease (e.g.,multiple myeloma-induced lytice bone disease), cystic fibrosis,age-related mascular degeneration (AMD; e.g., wet AMD and dry AMD),liver fibrosis, pulmonary fibrosis, atherosclerosis, cardiacischemia/reperfusion injury, heart failure, myocarditis, cardiacfibrosis, adverse myocardial remodeling, Guillain-Barre syndrome,Hashimoto's thyroiditis, psoriasis, psoritic arthritis, Crohn's Disease,ulcerative colitis, irritable bowel syndrome (IBS), inflammatory boweldisease (IBD), lupus nephritis, IgA nephropathy, diabetic kidneydisease, minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis (FSGS), nephrogenic systemic fibrosis (NSF),nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis,eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popularmucinosis), scleroderma, lichen sclerosusetatrophicus, POEMs syndrome(Crow-Fukase syndrome, Takatsuki disease or PEP syndrome), nephroticsyndrome, graft-versus-host-disease (GVHD), graft-versus-host-disease(GVHD) (from a transplant, such as blood, bone marrow, kidney, pancreas,liver, orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis). The bispecific antibody, antibody orantigen-binding fragment made be administered with anotherpharmaceutical agent, for example an anti-inflammatory agent, either inthe same formulation or separately.

In another embodiment, the present invention provides a compositioncomprising an antibody, e.g., a bispecific antibody, as described hereinand a pharmaceutically acceptable carrier. A pharmaceutical compositioncomprising an antibody, e.g., a bispecific antibody, of the inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the therapeutic proteins are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient patient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Gennaro, ed., Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing Company (1995).

For pharmaceutical use, an antibody, e.g., a bispecific antibody, of thepresent invention are formulated for parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.Intravenous administration may be by bolus injection, controlledrelease, e.g., using mini-pumps or other appropriate technology, or byinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include an antibody, e.g., a bispecificantibody, of the invention in combination with a pharmaceuticallyacceptable carrier, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. When utilizing such acombination therapy, the antibodies, which include bispecificantibodies, may be combined in a single formulation or may beadministered in separate formulations. Methods of formulation are wellknown in the art and are disclosed, for example, in Gennaro, ed.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa.(1990), which is incorporated herein by reference. Therapeutic doseswill generally be in the range of 0.1 to 100 mg/kg of patient weight perday, preferably 0.5-20 mg/kg per day, with the exact dose determined bythe clinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.More commonly, the antibodies will be administered over one week orless, often over a period of one to three days. Generally, the dosage ofadministered antibodies will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of antibodies which is in the range of fromabout 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient),although a lower or higher dosage also may be administered ascircumstances dictate.

Administration of an antibody, e.g., bispecific antibody, of theinvention to a subject can be intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic antibodies by injection, theadministration may be by continuous infusion or by single or multipleboluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase et al., “Oral Delivery of Microencapsulated Proteins”,in Sanders et al., eds., Protein Delivery: Physical Systems, pp.255-288, Plenum Press (1997)). The feasibility of an intranasal deliveryis exemplified by such a mode of insulin administration (see, forexample, Hinchcliffe et al., Adv. Drug Deliv. Rev., 35:199 (1999)). Dryor liquid particles comprising antibodies of the invention can beprepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit et al., TIBTECH, 16:343(1998); Patton et al., Adv. Drug Deliv. Rev., 35:235 (1999)). Thisapproach is illustrated by the AERX® diabetes management system, whichis a hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science, 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer a molecule having IL-17 and IL-23/p19 bindingactivity (Potts et al., Pharm. Biotechnol., 10:213 (1997)).

For purposes of therapy, compositions comprising an antibody, e.g., abispecific antibody, of the invention and a pharmaceutically acceptablecarrier are administered to a patient in a therapeutically effectiveamount. A combination of an antibody, e.g., a bispecific antibody, ofthe present invention and a pharmaceutically acceptable carrier is saidto be administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. For example, an agent used to treatinflammation is physiologically significant if its presence alleviatesthe inflammatory response. Effective treatment may be assessed in avariety of ways. In one embodiment, effective treatment is determined byreduced inflammation. In other embodiments, effective treatment ismarked by inhibition of inflammation. In still other embodiments,effective therapy is measured by increased well-being of the patientincluding such signs as weight gain, regained strength, decreased pain,thriving, and subjective indications from the patient of better health.

A pharmaceutical composition comprising an antibody, e.g., a bispecificantibody, of the invention can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.,10:239 (1997); Ranade, “Implants in Drug Delivery”, in Ranade et al.,eds., Drug Delivery Systems, pp. 95-123, CRC Press (1995); Bremer etal., “Protein Delivery with Infusion Pumps”, in Sanders et al., eds.,Protein Delivery: Physical Systems, pp. 239-254, Plenum Press (1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant”, in Sanders et al., eds., Protein Delivery: Physical Systems,pp. 93-117, Plenum Press (1997).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis., 12(Suppl. 1):561 (1993), Kim, Drugs, 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers”, inRanade et al., eds., Drug Delivery Systems, pp. 3-24, CRC Press (1995)).Liposomes are similar in composition to cellular membranes and as aresult, liposomes can be administered safely and are biodegradable.Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.02 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s) (see,for example, Machy et al., Liposomes in Cell Biology and Pharmacology,John Libbey (1987), and Ostro et al., American J. Hosp. Pharm., 46:1576(1989)). Moreover, it is possible to control the therapeuticavailability of the encapsulated agent by varying liposome size, thenumber of bilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

Liposomes can absorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci., 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta, 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta, 1068:133 (1991); Allen et al., Biochim. Biophys. Acta,1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent No. 04-244,018; Kato et al., Biol. Pharm. Bull.,16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull., 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato et al., Crit. Rev. Ther.Drug Carrier Syst., 14:287 (1997); Murahashi et al., Biol. Pharm. Bull.,20:259 (1997)). Similarly, Wu et al., Hepatology, 27:772 (1998), haveshown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull., 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA, 94:11681 (1997)).Moreover, Geho et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev., 32:99(1998)). After plasma elimination of free antibody,streptavidin-conjugated liposomes are administered. In another approach,targeting antibodies are directly attached to liposomes (Harasym et al.,Adv. Drug Deliv. Rev., 32:99 (1998)).

Antibodies can be encapsulated within liposomes using standardtechniques of protein microencapsulation (see, for example, Anderson etal., Infect. Immun., 31:1099 (1981), Anderson et al., Cancer Res.,50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta, 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies”, in Gregoriadis, ed., Liposome Technology, 2nd Edition, Vol.III, p. 317, CRC Press (1993), Wassef et al., Meth. Enzymol., 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta, 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz etal., Bioconjugate Chem., 6:332 (1995); Ranade, “Role of Polymers in DrugDelivery”, in Ranade et al., eds., Drug Delivery Systems, pp. 51-93, CRCPress (1995); Roskos et al., “Degradable Controlled Release SystemsUseful for Protein Delivery”, in Sanders et al., eds., Protein Delivery:Physical Systems, pp. 45-92, Plenum Press (1997); Bartus et al.,Science, 281:1161 (1998); Putney et al., Nature Biotechnology, 16:153(1998); Putney, Curr. Opin. Chem. Biol., 2:548 (1998)). Polyethyleneglycol (PEG)-coated nanospheres can also provide carriers forintravenous administration of therapeutic proteins (see, for example,Gref et al., Pharm. Biotechnol., 10:167 (1996).

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, an antibody, e.g., a bispecific antibody, of theinvention is administered in combination therapy, i.e., combined withother agents, e.g., therapeutic agents, that are useful for treatingpathological conditions or disorders, such as autoimmune disorders andinflammatory diseases. The term “in combination” in this context meansthat the agents are given substantially contemporaneously, eithersimultaneously or sequentially. If given sequentially, at the onset ofadministration of the second compound, the first of the two compounds ispreferably still detectable at effective concentrations at the site oftreatment.

For example, the combination therapy can include one or more anantibodies, e.g., bispecific antibodies, of the invention coformulatedwith, and/or coadministered with, one or more additional therapeuticagents, e.g., one or more cytokine and growth factor inhibitors,immunosuppressants, anti-inflammatory agents, metabolic inhibitors,enzyme inhibitors, and/or cytotoxic or cytostatic agents, as describedin more detail below. Furthermore, one or more antibodies, e.g.,bispecific antibodies, described herein may be used in combination withtwo or more of the therapeutic agents described herein. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

Preferred therapeutic agents used in combination with an antibody, e.g.,bispecific antibody, of the invention are those agents that interfere atdifferent stages in an inflammatory response. In one embodiment, one ormore antibodies, e.g., bispecific antibodies, described herein may becoformulated with, and/or coadministered with, one or more additionalagents such as other cytokine or growth factor antagonists (e.g.,soluble receptors, peptide inhibitors, small molecules, ligand fusions);or antibodies or antigen binding fragments thereof that bind to othertargets (e.g., antibodies that bind to other cytokines or growthfactors, their receptors, or other cell surface molecules); andanti-inflammatory cytokines or agonists thereof. Nonlimiting examples ofthe agents that can be used in combination with the antibodies describedherein, include, but are not limited to, antagonists of one or moreinterleukins (ILs) or their receptors, e.g., antagonists of IL-1, IL-2,IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-20, IL-21, IL-22and IL-31; antagonists of cytokines or growth factors or theirreceptors, such as tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGFand PDGF. Antibodies of the invention can also be combined withinhibitors of, e.g., antibodies to, cell surface molecules such as CD2,CD3, CD4, CD8, CD20 (e.g., the CD20 inhibitor rituximab (RITUXAN®)),CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, ortheir ligands, including CD154 (gp39 or CD40L), or LFA-1/ICAM-1 andVLA-4/VCAM-1 (Yusuf-Makagiansar et al., Med. Res. Rev., 22:146-167(2002)). Preferred antagonists that can be used in combination with oneor more antibodies, e.g., bispecific antibodies, described hereininclude antagonists of IL-1, IL-6, IL-12, TNF-alpha, IL-15, IL-18,IL-20, IL-22 and IL-31.

Examples of those agents include IL-12 antagonists, such as chimeric,humanized, human or in vitro-generated antibodies (or antigen bindingfragments thereof) that bind to IL-12 (preferably human IL-12), e.g.,the antibody disclosed in WO 00/56772; IL-12 receptor inhibitors, e.g.,antibodies to human IL-12 receptor; and soluble fragments of the IL-12receptor, e.g., human IL-12 receptor. Examples of IL-15 antagonistsinclude antibodies (or antigen binding fragments thereof) against IL-15or its receptor, e.g., chimeric, humanized, human or in vitro-generatedantibodies to human IL-15 or its receptor, soluble fragments of theIL-15 receptor, and IL-15-binding proteins. Examples of IL-18antagonists include antibodies, e.g., chimeric, humanized, human or invitro-generated antibodies (or antigen binding fragments thereof), tohuman IL-18, soluble fragments of the IL-18 receptor, and IL-18 bindingproteins (IL-18BP). Examples of IL-1 antagonists includeInterleukin-1-converting enzyme (ICE) inhibitors, such as Vx740, IL-1antagonists, e.g., IL-1 RA (anikinra, KINERET®, Amgen), sIL1RII(Immunex), and anti-IL-1 receptor antibodies (or antigen bindingfragments thereof).

Examples of TNF antagonists include chimeric, humanized, human or invitro-generated antibodies (or antigen binding fragments thereof) to TNF(e.g., human TNF-alpha), such as (HUMIRA®, D2E7, human TNF-alphaantibody), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNF-alphaantibody; Celltech/Pharmacia), cA2 (chimeric anti-TNF-alpha antibody;REMICADE®, Centocor); anti-TNF antibody fragments (e.g., CPD870);soluble fragments of the TNF receptors, e.g., p55 or p75 human TNFreceptors or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL®; Immunex), p55 kdTNFR-IgG (55 kDTNF receptor-IgG fusion protein (Lenercept)); enzyme antagonists, e.g.,TNF-alpha converting enzyme (TACE) inhibitors (e.g., an alpha-sulfonylhydroxamic acid derivative, and N-hydroxyformamide TACE inhibitor GW3333, -005, or -022); and TNF-bp/s-TNFR (soluble TNF binding protein).Preferred TNF antagonists are soluble fragments of the TNF receptors,e.g., p55 or p75 human TNF receptors or derivatives thereof, e.g., 75kdTNFR-IgG, and TNF-alpha converting enzyme (TACE) inhibitors.

In other embodiments, one or more antibodies, e.g., bispecificantibodies, described herein may be administered in combination with oneor more of the following: IL-13 antagonists, e.g., soluble IL-13receptors (sIL-13) and/or antibodies against IL-13; IL-2 antagonists,e.g., DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins, Seragen),and/or antibodies to IL-2R, e.g., anti-Tac (humanized anti-IL-2R,Protein Design Labs). Yet another combination includes one or moreantibodies, e.g., bispecific antibodies, of the invention, antagonisticsmall molecules, and/or inhibitory antibodies in combination withnondepleting anti-CD4 inhibitors (DEC-CE9.1/SB 210396; nondepletingprimatized anti-CD4 antibody; IDEC/SmithKline). Yet other preferredcombinations include antagonists of the costimulatory pathway CD80(B7.1) or CD86 (B7.2), including antibodies, soluble receptors orantagonistic ligands; as well as p-selectin glycoprotein ligand (PSGL),anti-inflammatory cytokines, e.g., IL-4 (DNAX/Schering); IL-10 (SCH52000; recombinant IL-10 DNAX/Schering); IL-13 and TGF-beta, andagonists thereof (e.g., agonist antibodies).

In other embodiments, one or more antibodies, e.g., bispecificantibodies, of the invention can be coformulated with, and/orcoadministered with, one or more anti-inflammatory drugs,immunosuppressants, or metabolic or enzymatic inhibitors. Nonlimitingexamples of the drugs or inhibitors that can be used in combination withthe antibodies described herein, include, but are not limited to, one ormore of: nonsteroidal anti-inflammatory drug(s) (NSAIDs), e.g.,ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, andindomethacin; sulfasalazine; corticosteroids such. as prednisolone;cytokine suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors ofnucleotide biosynthesis, e.g., inhibitors of purine biosynthesis, folateantagonists (e.g., methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotatedehydrogenase (DHODH) inhibitors. Preferred therapeutic agents for usein combination with one or more antibodies, e.g., bispecific antibodies,of the invention include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g.,leflunomide), and folate antagonists (e.g., methotrexate).

Examples of additional inhibitors include one or more of:corticosteroids (oral, inhaled and local injection); immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g.,sirolimus (rapamycin—RAPAMUNE® or rapamycin derivatives, e.g., solublerapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,CCI-779); agents which interfere with signaling by proinflammatorycytokines such as TNF-alpha or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAPkinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, andvariants thereof; phosphodiesterase inhibitors, e.g., R973401(phosphodiesterase Type IV inhibitor); phospholipase inhibitors, e.g.,inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethylketone analogs); inhibitors of vascular endothelial cell growth factoror growth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor;and inhibitors of angiogenesis. Preferred therapeutic agents for use incombination with the antibodies of the invention are immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus(rapamycin) or rapamycin derivatives, e.g., soluble rapamycinderivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2inhibitors, e.g., celecoxib and variants thereof; and phospholipaseinhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g.,trifluoromethyl ketone analogs.

Additional examples of therapeutic agents that can be combined with anantibody, e.g., bispecific antibody, of the invention include one ormore of: 6-mercaptopurines (6-MP); azathioprine sulphasalazine;mesalazine; olsalazine; chloroquine/hydroxychloroquine (PLAQUENIL®);pencillamine; aurothiornalate (intramuscular and oral); azathioprine;coichicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline,salmeteral); xanthines (theophylline, aminophylline); cromoglycate;nedocromil; ketotifen; ipratropium and oxitropium; mycophenolatemofetil; adenosine agonists; antithrombotic agents; complementinhibitors; and adrenergic agents.

Nonlimiting examples of agents for treating or preventing arthriticdisorders (e.g., rheumatoid arthritis, inflammatory arthritis,rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis andpsoriatic arthritis), with which an antibody, e.g., bispecific antibody,of the invention may be combined include one or more of the following:IL-12 antagonists as described herein; NSAIDs; CSAIDs; TNFs, e.g.,TNF-alpha, antagonists as described herein; nondepleting anti-CD4antibodies as described herein; IL-2 antagonists as described herein;anti-inflammatory cytokines, e.g., IL-4, IL-10, IL-13 and TGF-alpha, oragonists thereof; IL-1 or IL-1 receptor antagonists as described herein;phosphodiesterase inhibitors as described herein; Cox-2 inhibitors asdescribed herein; iloprost: methotrexate; thalidomide andthalidomide-related drugs (e.g., Celgen); leflunomide; inhibitor ofplasminogen activation, e.g., tranexamic acid; cytokine inhibitor, e.g.,T-614; prostaglandin E1; azathioprine; an inhibitor of interleukin-1converting enzyme (ICE); zap-70 and/or 1ck inhibitor (inhibitor of thetyrosine kinase zap-70 or 1ck); an inhibitor of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptoras described herein; an inhibitor of angiogenesis as described herein;corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-convertaseinhibitors; IL-11; IL-13; IL-17 inhibitors; gold; penicillamine;chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide;cyclosporine; total lymphoid irradiation; antithymocyte globulin;CD5-toxins; orally administered peptides and collagen; lobenzaritdisodium; cytokine regulating agents (CRAs) HP228 and HP466 (HoughtenPharmaceuticals, Inc.); ICAM-1 antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP 10; T Cell Sciences, Inc.); prednisone;orgotein; glycosaminoglycan polysulphate; minocycline (MINOCIN®);anti-IL2R antibodies; marine and botanical lipids (fish and plant seedfatty acids); auranofin; phenylbutazone; meclofenamic acid; flufenamicacid; intravenous immune globulin; zileuton; mycophenolic acid(RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose(therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.Preferred combinations include one or more antibodies, e.g., bispecificantibodies, of the invention in combination with methotrexate orleflunomide, and in moderate or severe rheumatoid arthritis cases,cyclosporine.

Preferred examples of inhibitors to use in combination with one or moreantibodies, e.g., bispecific antibodies, of the invention to treatarthritic disorders include TNF antagonists (e.g., chimeric, humanized,human or in vitro-generated antibodies, or antigen binding fragmentsthereof, that bind to TNF; soluble fragments of a TNF receptor, e.g.,p55 or p75 human TNF receptor or derivatives thereof, e.g., 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL®), p55 kD TNFreceptor-IgG fusion protein; TNF enzyme antagonists, e.g., TNF-alphaconverting enzyme (TACE) inhibitors); antagonists of IL-12, IL-15,IL-18, IL-22; T cell and B cell-depleting agents (e.g., anti-CD4 oranti-CD22 antibodies); small molecule inhibitors, e.g., methotrexate andleflunomide; sirolimus (rapamycin) and analogs thereof, e.g., CCI-779;cox-2 and cPLA2 inhibitors; NSAIDs; p38 inhibitors, TPL-2, Mk-2 and NFκBinhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g.,small molecule inhibitors, antibodies thereto, e.g., antibodies toP-selectin); estrogen receptor beta (ERB) agonists or ERB-NFκBantagonists. Most preferred additional therapeutic agents that can becoadministered and/or coformulated with one or more antibodies, e.g.,bispecific antibodies, of the invention include one or more of: asoluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptoror derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgGfusion protein, ENBREL®); methotrexate, leflunomide, or a sirolimus(rapamycin) or an analog thereof, e.g., CCI-779.

Nonlimiting examples of agents for treating or preventing multiplesclerosis with one or more antibodies, e.g., bispecific antibodies, ofthe invention can be combined include the following: interferons, e.g.,interferon-alpha1a (e.g., AVONEX®, Biogen) and interferon-1b(BETASERON®, Chiron/Berlex); Copolymer 1 (Cop-1; COPAXONE®, TevaPharmaceutical Industries, Inc.); dimethyl fumarate (e.g., BG-12;Biogen); hyperbaric oxygen; intravenous immunoglobulin; cladribine; TNFantagonists as described herein; corticosteroids; prednisolone;methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;cyclosporine A, methotrexate; 4-aminopyridine; and tizanidine.Additional antagonists that can be used in combination with antibodiesof the invention include antibodies to or antagonists of other humancytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6,EL-7, IL-8, IL-12 IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF.Antibodies as described herein can be combined with antibodies to cellsurface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40,CD45, CD69, CD80, CD86, CD90 or their ligands. One or more antibodies,e.g., bispecific antibodies, of the invention may also be combined withagents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signaling byproinflammatory cytokines as described herein, IL-1b converting enzymeinhibitors (e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, T-cellsignaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathloprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof, as described herein, and anti-inflammatory cytokines (e.g.,IL-4, IL-10, IL-13 and TGF).

Preferred examples of therapeutic agents for multiple sclerosis withwhich the antibodies of the invention can be combined include dimethylfumarate (e.g., BG-12; Biogen), interferon-beta, for example,IFN-beta-1a and IFN-beta-1b; COPAXONE®, corticosteroids, IL-1inhibitors, TNF inhibitors, antibodies to CD40 ligand and CD80, IL-12antagonists.

Nonlimiting examples of agents for treating or preventing inflammatorybowel disease (e.g., Crohn's disease, ulcerative colitis) with which anantibody, e.g., bispecific antibody, of the invention can be combinedinclude the following: budenoside; epidermal growth factor;corticosteroids; cyclosporine; sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonal antibodies;anti-IL-6 monoclonal antibodies (e.g., anti-IL-6 receptor antibodies andanti-IL-6 antibodies); growth factors; elastase inhibitors;pyridinyl-imidazole compounds; TNF antagonists as described herein;IL-4, IL-10, IL-13 and/or TGF.beta. cytokines or agonists thereof (e.g.,agonist antibodies); IL-11; glucuronide- or dextran-conjugated prodrugsof prednisolone, dexamethasone or budesonide; ICAM-1 antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals,Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);slow-release mesalazine; methotrexate; antagonists of plateletactivating factor (PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of agents for treating or preventing psoriasis withwhich an antibody, e.g., bispecific antibody, of the invention can becombined include the following: corticosteroids; vitamin D₃ and analogsthereof; retinoiods (e.g., soriatane); methotrexate; cyclosporine,6-thioguanine; Accutane; hydrea; hydroxyurea; sulfasalazine;mycophenolate mofetil; azathioprine; tacrolimus; fumaric acid esters;biologics such as AMEVIVE®, ENBREL®, HUMIRA®, Raptiva and REMICADE®,Ustekinmab, and XP-828L; phototherapy; and photochemotherapy (e.g.,psoralen and ultraviolet phototherapy combined).

Nonlimiting examples of agents for treating or preventing inflammatoryairway/respiratory disease (e.g., chronic obstructive pulmonarydisorder, asthma) with which an antibody, e.g., bispecific antibody, ofthe invention can be combined include the following: beta2-adrenoceptoragonists (e.g., salbutamol (albuterol USAN), levalbuterol, terbutaline,bitolterol); long-acting beta2-adrenoceptor agonists (e.g., salmeterol,formoterol, bambuterol); adrenergic agonists (e.g., inhaled epinephrineand ephedrine tablets); anticholinergic medications (e.g., ipratropiumbromide); combinations of inhaled steroids and long-actingbronchodilators (e.g., fluticasone/salmeterol (ADVAIR® in the UnitedStates, and Seretide in the United Kingdom)) or. budesonide/formoterol(SYMBICORT®)); inhaled glucocorticoids (e.g., ciclesonide,beclomethasone, budesonide, flunisolide, fluticasone, mometasone,triamcinolone); leukotriene modifiers (e.g., montelukast, zafirlukast,pranlukast, and zileuton); mast cell stabilizers (e.g., cromoglicate(cromolyn), and nedocromil); antimuscarinics/anticholinergics (e.g.,ipratropium, oxitropium, tiotropium); methylxanthines (e.g.,theophylline, aminophylline); antihistamines; IgE blockers (e.g.,Omalizumab); M.sub.3 muscarinic antagonists (anticholinergics) (e.g.,ipratropium, tiotropium); cromones (e.g., chromoglicate, nedocromil);zanthines (e.g., theophylline); and TNF antagonists (e.g., infliximab,adalimumab and etanercept).

In one embodiment, an antibody, e.g., bispecific antibody, of theinvention can be used in combination with one or more antibodiesdirected at other targets involved in regulating immune responses, e.g.,transplant rejection.

Nonlimiting examples of agents for treating or preventing immuneresponses with which an antibody, e.g., bispecific antibody, of theinvention can be combined include the following: antibodies againstother cell surface molecules, including but not limited to CD25(interleukin-2 receptor-a), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45,CD28/CTLA4 (CD80 (B7.1), e.g., CTLA4 Ig-abatacept (ORENCIA®)), ICOSL,ICOS and/or CD86 (B7.2). In yet another embodiment, an antibody of theinvention is used in combination with one or more generalimmunosuppressive agents, such as cyclosporin A or FK506.

In other embodiments, antibodies are used as vaccine adjuvants againstautoimmune disorders, inflammatory diseases, etc. The combination ofadjuvants for treatment of these types of disorders are suitable for usein combination with a wide variety of antigens from targetedself-antigens, i.e., autoantigens, involved in autoimmunity, e.g.,myelin basic protein; inflammatory self-antigens, e.g., amyloid peptideprotein, or transplant antigens, e.g., alloantigens. The antigen maycomprise peptides or polypeptides derived from proteins, as well asfragments of any of the following: saccharides, proteins,polynucleotides or oligonucleotides, autoantigens, amyloid peptideprotein, transplant antigens, allergens, or other macromolecularcomponents. In some instances, more than one antigen is included in theantigenic composition.

For example, desirable vaccines for moderating responses to allergens ina vertebrate host, which contain the adjuvant combinations of thisinvention, include those containing an allergen or fragment thereof.Examples of such allergens are described in U.S. Pat. No. 5,830,877 andPCT Publication No. WO 99/51259, which are hereby incorporated byreference in their entireties, and include pollen, insect venoms, animaldander, fungal spores and drugs (such as penicillin). The vaccinesinterfere with the production of IgE antibodies, a known cause ofallergic reactions. In another example, desirable vaccines forpreventing or treating disease characterized by amyloid deposition in avertebrate host, which contain the adjuvant combinations of thisinvention, include those containing portions of amyloid peptide protein(APP). This disease is referred to variously as Alzheimer's disease,amyloidosis or amyloidogenic disease. Thus, the vaccines of thisinvention include the adjuvant combinations of this invention plus Aβpeptide, as well as fragments of Aβ peptide and antibodies to Aβ peptideor fragments thereof.

In another embodiment, pharmaceutical compositions may be supplied as akit comprising a container that comprises an antibody, bispecificantibody or antigen-binding fragment of the invention. Antibodies, e.g.,bispecific antibodies, of the invention can be provided in the form ofan injectable solution for single or multiple doses, or as a sterilepowder that will be reconstituted before injection. Alternatively, sucha kit can include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of the antibody, e.g., bispecific antibody.Such a kit may further comprise written information on indications andusage of the pharmaceutical composition. Moreover, such information mayinclude a statement that the antibody composition is contraindicated inpatients with known hypersensitivity to IL-17 and IL-23.

In a further embodiment, the invention provides an article ofmanufacture, comprising: (a) a composition of matter comprising anantibody, bispecific antibody or antigen-binding fragment as describedherein; (b) a container containing said composition; and (c) a labelaffixed to said container, or a package insert included in saidcontainer referring to the use of said antibody in the treatment of animmune related disease.

In another aspect, the composition comprises a further activeingredient, which may, for example, be a further antibody or ananti-inflammatory, cytotoxic or chemotherapeutic agent. Preferably, thecomposition is sterile.

The antibodies, bispecific antibodies and antigen-binding fragments asdescribed herein are also useful to prepare medicines and medicamentsfor the treatment of immune-related and inflammatory diseases, includingfor example, multiple sclerosis (MS), irritable bowel syndrome (IBS),inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn'sdisease, atopic dermatitis, contact dermatitis, systemic sclerosis,systemic lupus erythematosus (SLE), antineutrophil cytoplasmicantibodies (ANCA)-associated vasculitis (AAV), giant cell arteritis,multiple sclerosis (MS), colitis, endotoxemia, arthritis, rheumatoidarthritis (RA), osteoarthritis, Sjögren's syndrome, psoriasis, psoriaticarthritis, adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as idiopathic pulmonary fibrosis,asthma, chronic obstructive pulmonary disease (COPD), airwayhyper-responsiveness, chronic bronchitis, allergic asthma, eczema,Helicobacter pylori infection, intraabdominal adhesions and/or abscessesas results of peritoneal inflammation (e.g., from infection, injury,etc.), nephrotic syndrome, idiopathic demyelinating polyneuropathy,Guillain-Barre syndrome, organ allograft rejection, graft vs. hostdisease (GVHD), lupus nephritis, IgA nephropathy, diabetic kidneydisease, minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis (FSGS), nephrogenic systemic fibrosis (NSF),nephrogenic fibrosing dermopathy, fibrosing cholestatic hepatitis,eosinophilic fasciitis (Shulman's syndrome), scleromyxedema (popularmucinosis), scleroderma, lichen sclerosusetatrophicus, POEMs syndrome(Crow-Fukase syndrome, Takatsuki disease or PEP syndrome), nephroticsyndrome, graft-versus-host-disease (GVHD), graft-versus-host-disease(GVHD) (from a transplant, such as blood, bone marrow, kidney, pancreas,liver, orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), lytic bone disease (e.g., multiplemyeloma-induced lytice bone disease), cystic fibrosis, age-relatedmascular degeneration (AMD; e.g., wet AMD and dry AMD), liver fibrosis,pulmonary fibrosis, atherosclerosis, cardiac ischemia/reperfusioninjury, heart failure, myocarditis, cardiac fibrosis, adverse myocardialremodeling, transplant rejection, streptococcal cell wall (SCW)-inducedarthritis, gingivitis/periodontitis, herpetic stromal keratitis,gluten-sensitive enteropathy restenosis, Kawasaki disease, and immunemediated renal diseases. In a specific aspect, such medicines andmedicaments comprise a therapeutically effective amount of a bispecificantibody, antibody or antigen-binding fragment of the invention with apharmaceutically acceptable carrier. In an embodiment, the admixture issterile.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GENBANK®amino acid and nucleotide sequence submissions) cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Humanization of a Murine Anti-Human IL-17A/F Dual SpecificAntibody

Selection of Hybridoma Clones and Variable Region Identification

Recombinant human proteins IL-17A, IL-17A/F, and IL-17F were producedusing an HEK293 transient expression system at ZymoGenetics Inc., aBristol-Myers Squibb Company (Seattle, Wash., USA). BALB/c mice (CharlesRiver Laboratories, Wilmington, Mass.) were immunized and boosted withrecombinant human IL-17F conjugated to BSA followed by immunizationswith recombinant human IL-17A conjugated to BSA. The mice with seracontaining the highest anti-IL-17F and anti-IL-17A antibody bindingactivity were given a final pre-fusion boost of IL-17F. Four days later,the splenocytes and lymph node cells were fused with Ag8.653 myelomacells to generate antibody producing hybridomas. Hybridoma culturesupernatants were screened for IL-17F and IL-17A binding by plate basedELISA and IL-17F and IL-17A neutralization in the IL-17A/F cell-basedassay. Hybridoma cells corresponding to the supernatant sample thatbound and neutralized both IL-17F and IL-17A were cloned in order toisolate a monoclonal hybridoma, 339.15.3.5 (designated as 339.15)producing the neutralizing monoclonal antibody of interest. Hybridoma339.15 was isotyped using the ISOSTRIP® Mouse Monoclonal AntibodyIsotyping Kit (Roche, Indianapolis, Ind., USA) and RNA was isolatedusing the QIAGEN® RNeasy kit (Qiagen, Valencia, Calif., USA). Variableregions were cloned using the SMART RACE cDNA Amplification Kit(Clontech, Mountain View, Calif., USA), utilizing 5′ RACE technology andgene specific 3′ primers designed to mouse constant region sequences.Heavy and light variable region sequences were cloned using the TOPO® TACloning Kit for Sequencing (Invitrogen, Carlsbad, Calif., USA). Genesequences were verified by comparing the sequence to the N-terminalamino acid sequencing performed on antibody purified from hybridoma339.15.

Variable region sequences were cloned from 339.15.3.5 and 339.15.3.6 andshown to contain the same variable region sequences. The sequence from339.15.3.5 was used for subsequent humanization, and the 339.15.3.6hybridoma clone was deposited on Nov. 7, 2006, with the American TypeTissue Culture Collection (ATCC, 10801 University Blvd, Manassas, Va.20110-2209) patent depository as original deposits under the BudapestTreaty and was given ATCC® Patent Deposit Designation PTA-7988.Hybridoma clone 339.15.3.6 (ATCC® Patent Deposit Designation PTA-7988)is also disclosed, for example, in U.S. Pat. Nos. 7,790,163, 7,910,703and 8,333,968.

Molecular Modeling of Chimeric and Humanized Anti-Human IL-17A/FVariable Region Sequences

All variable region models were constructed and viewed using the MOESoftware Suite, Version 2008 (Chemical Computing Group, Montreal,Canada).

Anti-Human IL-17A/F Humanized Antibody Design

Murine complementarity determining regions (CDR) were grafted onto humangermline framework sequences. The sequences were compared to germlineamino acid sequences in V-Base (MRC, Center for Protein Engineering,UK). One germline gene was chosen for the variable heavy region, VH1-03.Several germline genes were chosen for the variable light region; VKVIA26, VKI A20, VKVI A14, VKIII L6, and VKI L14. The VKVI germline genefamily showed the highest homology to the murine sequence, however,being an under represented germline family in the human antibodyrepertoire, other germline families with high homology were alsoconsidered. Murine Kabat defined CDR regions were grafted on human Kabatdefined framework regions for both the heavy and light chains.

Construction, Expression, and Purification of Humanized Anti-IL-17A/FAntibodies

Humanized variable region sequences were ordered from GeneART, Inc.(GeneART, Inc. Burlingame, Calif., USA). Humanized and murine variableregion sequences were fused to human kappa constant region (SEQ IDNO:10) or IgG1.1 (SEQ ID NO:11), an effector minus variant of wild-typeIgG1 that has mutations resulting in the reduction of Fc γ receptor Ibinding and ability to fix complement (Gross et al., Immunity,15:289-302 (2001)), utilizing overlap PCR (Horton et al., Gene, 77:61-68(1989)) and/or restriction enzyme cloning into pTT5, an HEK293-6Etransient expression vector (NCR Biotechnology Research Institute,Ottawa, ON, CAN). All constructs were expressed using the mod2610(ATGCGGCGGAGAGGCTGGTCCTGGATCTTCCTGTTTCTGCTGAGCGGAACAG CCGGCGTGCTGAGC,SEQ ID NO:30) signal sequence, although any nucleic acid sequence thatencodes the amino acid sequence MRRRGWSWIFLFLLSGTAGVLS (SEQ ID NO:31)may be used. The HEK293-6E suspension cells were transfected withexpression constructs using polyethylenimine reagent and cultivated inF17 medium (Invitrogen, Grand Island, N.Y., USA) with the addition of 5mM L-glutamine and 25 μg/mL G418. After 24 hours, 1/40th volume of 20%Tryptone NI (Organotechnie SAS, La Courneuve, FR) was added. Atapproximately 120 hours post transfection, conditioned media washarvested and passed through a 0.2 μm filter. Protein was purified fromthe filtered conditioned media using a combination of Mab Select SuReAffinity Chromatography (GE Healthcare, Piscataway, N.J., USA) andSUPERDEX® 200 Size Exclusion Chromatography (GE Healthcare, Piscataway,N.J., USA). Content was estimated by absorbance at UV-A280 nm andquality evaluated by analytical size exclusion high performance liquidchromatography, SDS PAGE, and western blot.

Anti-Human IL-17A/F Humanization Panel Bioassay Activity; NIH/3T3/KZ170NF-κB Luciferase Reporter Assay to Measure Human IL-17A, IL-17A/F, andIL-17F Activity by NE-κB Induction

A murine fibroblast cell line (NIH/3T3, ATCC® #CRL-1658) was stablytransfected with an NF-κB luciferase reporter designated KZ170 andcloned out. NIH/3T3/KZ170 clone 1 cells were seeded at 10,000 cells/wellin plating media (DMEM plus 3% FBS, 1 mM sodium pyruvate, 2 mML-glutamine (HyClone Laboratories, South Logan, Utah)) in 96-well, whiteopaque, solid bottom luciferase plates (Corning Incorporated, Corning,N.Y.) and incubated overnight at 37° C., 5% CO₂. The following dayserial dilutions of recombinant human IL-17A, IL-17A/F, or IL-17F(ZymoGenetics, A Bristol-Myers Squibb Company, Seattle, Wash., USA) weremade up in assay media (DMEM plus 0.5% BSA, 1 mM sodium pyruvate, 2 mML-glutamine, 10 mM HEPES (HyClone Laboratories, South Logan, Utah)) andadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 4 hours. Additionally the assay was used to measureneutralization of human IL-17A, IL-17A/F and IL-17F activity. A halfmaximal concentration (EC₅₀, effective concentration at 50 percent) ofhuman IL-17A, IL-17A/F or IL-17F was combined with serial dilutions ofanti-human IL-17A/F antibodies described herein in assay media and addedto the plates containing the cells and incubated together at 37° C., 5%CO₂ for 4 hours. Following incubation the media was removed and cellslysed before being read on the Berthold Centro XS³ Luminometer (BertholdTechnologies, Wildbad, Germany) using flash substrate (PromegaCorporation, Madison, Wis.) according to manufacturer's instructions.Increases in mean fluorescence intensity (via activation of the NF-κBluciferase reporter) were indicative of a human IL-17A, IL-17A/F, IL-17Freceptor-ligand interaction. Decreases in mean fluorescence intensitywere indicative of neutralization of the human IL-17A, IL-17A/F, IL-17Freceptor-ligand interaction. IC₅₀ (inhibitory concentration at 50percent) values were calculated using GraphPad Prism 4 software(GraphPad Software, Inc., San Diego Calif.) for each anti-human IL-17A/Fantibody.

Anti-Human IL-17A/F Humanization CDR Grafted and Chimeric Panel BioassayActivity; NIH/3T3/KZ170 NF-κB Luciferase Reporter Assay Results

IL-17A, IL-17A/F and IL-17F induce activation of the NF-κB luciferasereporter in a dose dependent manner with an EC₅₀ concentrationdetermined to be 0.15 nM for IL-17A, 0.50 nM for IL-17A/F and 0.50 nMfor IL-17F. Tables 1 and 2 present example IC₅₀ data for theanti-IL-17A/F antibodies described herein.

TABLE 1 IL- VH VL IL- 17F MVC# MVC# IL-17A 17A/F IC₅₀ Name SEQ ID NO:SEQ ID NO: IC₅₀ nM IC₅₀ nM nM Chi- Ms VH VR370 Ms VL VR371 11 0.30 0.26meric MVC823 MVC824 339.15 SEQ ID NO: 32 SEQ ID NO: 34 339-07  VR370e3VH1- Ms VL >600 31 5.5 03 MVC824 MVC840 SEQ ID NO: 34 SEQ ID NO: 36339-08  Ms VH VR370 VR371e3 VKVI 1.5 0.96 0.81 MVC823 A26 SEQ ID NO: 32MVC841 SEQ ID NO: 38 339-02  Ms VH VR370 VR371e2 VKI 1.1 0.80 0.79MVC823 A20 SEQ ID NO: 32 MVC717 SEQ ID NO: 40 339-01  Ms VH VR370VR371e1 VKVI 9.6 0.26 0.20 MVC823 A14 SEQ ID NO: 32 MVC716 SEQ ID NO: 42339-09  Ms VH VR370 VR371e4 VKIII 7.2 0.20 0.21 MVC823 L6 SEQ ID NO: 32MVC842 SEQ ID NO: 44 339-32  Ms VH VR370 VR371e10 VKI 7.0 1.5 0.35MVC823 L14 SEQ ID NO: 32 MVC856 SEQ ID NO: 46 339-33  VR370e3 VH1-VR371e3 VKVI >600 9.7 1.7 03 A26 MVC840 MVC841 SEQ ID NO: 36 SEQ ID NO:38 339-126 VR370e3 VH1- VR371e2 VKI >600 24 1.6 03 A20 MVC840 MVC717 SEQID NO: 36 SEQ ID NO: 40Anti-Human IL-17A/F Humanization CDR Grafted with Framework BackMutation Panel Bioassay Activity Table: NIH/3T3/KZ170 NF-κB LuciferaseReporter Assay Results

TABLE 2 IL- VH VL IL- 17F MVC# MVC# IL-17A 17A/F IC₅₀ Name SEQ ID NO:SEQ ID NO: IC₅₀ nM IC₅₀ nM nM 339-35  VR370e4 NKSH VR371e3 VKVI >600 2.40.64 MVC850 A26 SEQ ID NO: 48 MVC841 SEQ ID NO: 38 339-71  VR370e41 KALVVR371e3 VKVI 16 0.37 0.20 MVC869 A26 SEQ ID NO: 50 MVC841 SEQ ID NO: 38339-37  VR370e6 SF VR371e3 VKVI >600 20 3.5 MVC852 A26 SEQ ID NO: 52MVC841 SEQ ID NO: 38 339-38  VR370e7 VR371e3 VKVI 8.2 0.27 0.27 NKSHKALV A26 MVC853 MVC841 SEQ ID NO: 54 SEQ ID NO: 38 339-39  VR370e8VR371e3 VKVI 7.6 0.23 0.25 NKSH KALV SF A26 MVC854 MVC841 SEQ ID NO: 56SEQ ID NO: 38 339-127 VR370e4 NKSH VR371e2 VKI 190 3.4 0.50 MVC850 A20SEQ ID NO: 48 MVC717 SEQ ID NO: 40 339-128 VR370e41 KALV VR371e2 VKI 5.10.41 0.25 MVC869 A20 SEQ ID NO: 50 MVC717 SEQ ID NO: 40 339-105 VR370e6SF VR371e2 VKI >600 23 2.6 MVC852 A20 SEQ ID NO: 52 MVC717 SEQ ID NO: 40339-125 VR370e7 VR371e2 VKI 1.5 0.81 0.83 NKSH KALV A20 MVC853 MVC717SEQ ID NO: 54 SEQ ID NO: 40 339-104 VR370e8 VR371e2 VKI 1.5 0.83 0.83NKSH KALV SF A20 MVC854 MVC717 SEQ ID NO: 56 SEQ ID NO: 40 339-134VR370e96 VR371e2 VKI 1.4 0.26 0.24 NK KALV A20 MVC978 MVC717 SEQ ID NO:58 SEQ ID NO: 40Anti-Human IL-17A/F Humanization Panel Biacore Activity; Measurement ofBinding Affinities to Human IL-17A, IL-17A/F, and IL-17F Via SurfacePlasmon Resonance (Biacore)

Humanized anti-human IL-17A/F monoclonal antibodies were evaluated fortheir binding affinity to human IL-17A, human IL-17A/F, and human IL-17Fusing surface plasmon resonance.

Kinetic rate constants and equilibrium dissociation constants weremeasured for the interaction of the humanized anti-human IL-17A/Fantibodies with human IL-17A, IL-17A/F, and IL-17F via surface plasmonresonance. The association rate constant (k_(a) (M⁻¹s⁻¹)) is a valuethat reflects the rate of the antigen-antibody complex formation. Thedissociation rate constant (k_(d) (s⁻¹)) is a value that reflects thestability of this complex. By dividing the dissociation rate constant bythe association rate constant (k_(d)/k_(a)) the equilibrium dissociationconstant (K_(D) (M)) is obtained. This value describes the bindingaffinity of the interaction. Antibodies with similar K_(D) can havewidely variable association and dissociation rate constants.Consequently, measuring both the k_(a) and k_(d) of antibodies helps tomore uniquely describe the affinity of the antibody-antigen interaction.

Binding kinetics and affinity studies were performed on a BIACORE® T100system (GE Healthcare, Piscataway, N.J.). Methods for the BIACORE® T100were programmed using BIACORE® T100 Control Software, v 2.0. For theseexperiments, the humanized anti-human IL-17A/F antibodies were capturedonto a CM4 sensor chip via either goat anti-human IgG Fc-gamma antibody(Jackson ImmunoResearch, West Grove, Pa.) or goat anti-mouse IgGFc-gamma antibody (Jackson ImmunoResearch). Binding experiments with theIL-17 molecules were performed at 25° C. in a buffer of 10 mM HEPES, 150mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GE Healthcare), 1 mg/mL bovineserum albumin, pH 7.4.

The capture antibody, goat anti-human IgG Fc-gamma, was diluted toconcentration of 20 μg/mL in 10 mM sodium acetate pH 5.0, and thencovalently immobilized to all four flow cells of a CM4 sensor chip usingamine coupling chemistry (EDC:NHS). After immobilization of theantibody, the remaining active sites on the flow cell were blocked with1 M ethanolamine. A capture antibody density of approximately 5000 RUwas obtained. The humanized anti-human IL-17A/F antibodies were capturedonto flow cell 2, 3, or 4 of the CM4 chip at a density ranging from60-150 RU. Capture of the test antibodies to the immobilized surface wasperformed at a flow rate of 10 μL/min. The BIACORE® instrument measuresthe mass of protein bound to the sensor chip surface, and thus, captureof the test antibody was verified for each cycle. Serial dilutions ofhuman IL-17A, IL-17A/F, or IL-17F (ZymoGenetics, A Bristol-Myers, SquibbCompany, Seattle, Wash., USA) were prepared from 100 nM-0.032 nM (1:5serial dilutions). The serial dilutions were injected over the surfaceand allowed to specifically bind to the test antibody captured on thesensor chip. Duplicate injections of each antigen concentration wereperformed with an association time of 7 minutes and dissociation time of15 minutes. Kinetic binding studies were performed with a flow rate of50 μL/min. In between cycles, the flow cell was washed with 20 mMhydrochloric acid to regenerate the surface. This wash step removed boththe captured test antibody and any bound antigen from the immobilizedantibody surface. The test antibody was subsequently captured again inthe next cycle.

Data was compiled using the BIACORE® T100 Evaluation software (version2.0). Data was processed by subtracting reference flow cell and blankinjections. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Duplicate injection curves were checked forreproducibility. Based on the binding of the bivalent IL-17 molecules toa bivalent antibody, the bivalent analyte binding interaction model wasdetermined to be appropriate for interactions with the IL-17 molecules.The bivalent analyte model is previously described in West, A. P. etal., Biochemistry, 39:9698-9708 (2000); and West, A. P. et al., J. Mol.Biol., 313:385-397 (2001). An affinity constant (K_(D1)) under thebivalent analyte model may be calculated from the ratio of rateconstants (k_(d1)/k_(a1)) as determined by surface plasmon resonance.The reference subtracted binding curves were globally fit to theappropriate binding model with a multiple Rmax and with the RI set tozero. The data fit well to the binding models with good agreementbetween the experimental and theoretical binding curves. The chi² andstandard errors associated the fits were low. There was no trending inthe residuals.

Anti-Human IL-17A/F Humanization Panel Biacore Activity

The results of the binding experiments with human IL-17A, IL-17A/F, andIL-17F are in Tables 3, 4, and 5 respectively.

Anti-Human IL-17A/F Humanized Antibodies Binding Affinity for IL-17A

TABLE 3 k_(a1) k_(d1) Name (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M) Mouse 4.E+06 7.E−032.E−09 339.15 339-02 2.E+06 6.E−03 3.E−09 SEQ ID NO: 32 SEQ ID NO: 40Chimeric 3.E+06 1.E−02 4.E−09 339.15 SEQ ID NO: 32 SEQ ID NO: 34 339-384.E+06 5.E−03 1.E−9 SEQ ID NO: 54 SEQ ID NO: 38 339-125 2.E+06 3.E−031.E−9 SEQ ID NO: 54 SEQ ID NO: 40 339-134 2.E+06 3.E−03 1.E−9 SEQ ID NO:58 SEQ ID NO: 40Anti-IL-17A/F Humanized Antibodies Binding Affinity for IL-17A/F

TABLE 4 k_(a1) k_(d1) Name (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M) Mouse 1.E+06 2.E−042.E−10 339.15 339-02 Not Determined SEQ ID NO: 32 SEQ ID NO: 40 ChimericNot Determined 339.15 SEQ ID NO: 32 SEQ ID NO: 33 339-38 1.E+06 4.E−044.E−10 SEQ ID NO: 54 SEQ ID NO: 40 339-125 2.E+06 6.E−04 3.E−10 SEQ IDNO: 54 SEQ ID NO: 40 339-134 2.E+06 5.E−04 2.E−10 SEQ ID NO: 58 SEQ IDNO: 40Anti-IL-17A/F Humanized Antibodies Binding Affinity for IL-17F

TABLE 5 k_(a1) k_(d1) Name (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M) Mouse 2.E+06 1.E−045.E−11 339.15 339-02 1.E+06 5.E−04 4E−10 SEQ ID NO: 32 SEQ ID NO: 40Chimeric 1.E+06 5.E−04 4E−10 339.15 SEQ ID NO: 32 SEQ ID NO: 34 339-382.E+06 6.E−04 3.E−10 SEQ ID NO: 54 SEQ ID NO: 40 339-125 2.E+06 2.E−041.E−10 SEQ ID NO: 54 SEQ ID NO: 40 339-134 2.E+06 2.E−04 1.E−10 SEQ IDNO: 58 SEQ ID NO: 40

EXAMPLE 2 7B7 Antibody Selection and Hybridoma Generation

Epitope Binning Approach by Surface Plasmon Resonance Technology (UsingBiacore) to Group Antibodies Based on their Binding and BlockingProperties as Shown in FIG. 11.

Antibodies were grouped and selected based on their ability to:

1. Specifically bind p19 subdomain only of IL-23;

2. Specifically block only IL-23 receptor (IL-23R) and not block IL-12receptor (IL-12R); and

3. Not compete with any antibody that could bind specifically to p40subdomain of IL-23.

Materials such as antibodies with previously known selectivity for p19or p40 subdomains of IL-23 and IL-23R or IL-12R were all chosen to becoated on a BIACORE® CM5 chip. The coating density varied between 500 to8000 Resonance Units (RUs). Antibodies that were to be binned weretitrated serially (1:2 or 1:3) to 8 concentrations, from startingconcentrations that ranged from 10 to 100 μg/mL in a 96-well ELISAplate. To each of the well, 10 nM of IL-23 antigen was added. Theantibodies on plate were allowed to form a complex with antigen andreach equilibrium overnight at 4° C. The complexes were injected overthe CM5 chip at a flow rate of 20 μL/min for two minutes. The signal, asbinding resonance units (RUs) at end of two minutes was noted. Theantibody-antigen complex was able complete or not compete with thematerial that was coated on the chip. If the antibody in complex withantigen was able to compete with the material on chip, with increasingconcentration of the antibody, the binding RU decreased and if it didnot compete, the binding RU increased. Based on this observation, allanti-IL23 antibodies were binned according to their bindingselectivities and competing abilities.

Transgenic HCo12 J/K HUMAB® Mice from the Medarex Colonies in Milpitas,Calif. were Immunized with Recombinant Human IL-23-His in RIBI Adjuvant.

Sera from immunized mice were tested for expression of IL-23 specificantibodies by a modified indirect dual ELISA. Briefly, microtiter plates(COSTAR®, 96-well flat bottom, #9018) were coated with mouse anti-hisprotein at 2.5 μg/ml in PBS, 50 μl/well, incubated at 4° C. overnight,and then blocked with 1% BSA in PBS. HuIL-23 at 2.5 μg/ml or HuIL-12 wasadded to plates for capture at 50 μl/well and incubated at roomtemperature for one hour. Plates were washed with PBS Tween, anddilutions of sera were added and incubated for 1 hour. The plates werewashed with PBS-Tween and incubated with goat-anti-human gamma heavychain conjugated with HRP (Jackson ImmunoResearch Cat. 109-036-098) for1 hour. After 3× washing, the plates were developed with ABTS (Moss, CAT#ABTS-1000) substrate and OD's analyzed at 415 nm. Data were analyzedand expressed as serum titer which is defined as the highest dilution ofserum which results in an antigen positive signal of at least twicebackground. Mouse 215094 was selected for hybridoma generation basedupon relatively high titers on IL-23 with lower cross reactivity toIL-12 when compared to other mice in the cohort (see Table 6).

TABLE 6 Serum Titers Mouse ID Genotype Hu IL23-his Hu IL12-his 215088HCo12:01[J/K] >109, 350 >109, 350 215090 HCo12:01[J/K] >109, 350 >109,350 215092 HCo12:01[J/K] >109, 350 >109, 350 215094 HCo12:01[J/K] >109,350  12, 150 215096 HCo12:01[J/K] >109, 350  36, 450 215098HCo12:01[J/K]  36, 450   1, 350 215089 HCo12:01[J/K] >109, 350   1, 350215091 HCo12:01[J/K] >109, 350 >109, 350 215093 HCo12:01[J/K] >109, 350  4, 050 215095 HCo12:01[J/K] >109, 350 >109, 350 215097HCo12:01[J/K] >109, 350 >109, 350 215099 HCo12:01[J/K] 12150  12, 150

The genotype of Mouse 215094 is provided below in Table 7.

TABLE 7 Mouse 215094 Genotype Mouse ID Sex Date of birth Genotype 215094M Oct. 11, 2009 HCo12(15087)+{circumflex over ( )}; JHD++; JKD++;KCo5(9272)+{circumflex over ( )};

The spleen from mouse 215094 was used to generate hybridomas with mousemyeloma cells (ATCC CRL-1580) by electric field based electrofusionusing a CytoPulse large chamber cell fusion electroporation device in aprocedure designated fusion 2378.

Conditioned media from the resulting hybridomas were initially screenedfor expression of human IgG γ/κ in a standard automated assay followedby ELISA for IL-23 binding with a counter screen ELISA on IL-12 toidentify specific clones as previously described. Hybridoma selectioncriteria for testing were samples with OD's greater than 1.5 on huIL23plates and less than 0.15 on huIL12.

Fusion 2378 generated total of 827 human IgG positive hybridomas ofwhich, 128 were IL-23 specific. Hybridoma 7B7 was selected for furthertesting based on its strong binding to IL-23 and lack of crossreactivity to IL-12, when compared to anti-p19 and anti-p40 positivecontrol antibodies; an example of hybridomas, including 7B7 selected byELISA is given in FIG. 7. The isotype of subclone 7B7.D4 was confirmedas human IgG1, kappa by ELISA.

Hybridoma conditioned medium from all IL-23p-19 specific MAbs werescreened for IL-23 neutralizing activity in a cell-based assay. Kit225,a human T-cell line established from a patient with T-cell chroniclymphocytic leukemia, have been shown to respond to IL-23 with dosedependant STAT3 phosphorylation (pSTAT3). Human IL-23 at EC₅₀ with andwithout the addition of hybridoma conditioned medium or a controlneutralizing anti-p19 antibody was used to stimulate cells for 15minutes. Cells were lysed and inhibition of IL-23 dependant STAT3phosphorylation was assessed by ELISA (Cell Signaling Technology,PATHSCAN® Cat #7300) where reduced O.D. indicates reduced levels ofpSTAT3. Hybridoma 7B7 was selected for sub-cloning and furthercharacterization based upon the potent neutralization of IL-23 signalingobserved in the Kit225 assay and as shown in FIG. 8.

Using assays similar to those described above, selective binding ofIL-23 and neutralization of IL-23 signaling was demonstrated for the 7B7subclone 1413.2378.7B7.D4.H2 which was subsequently submitted forsequencing (IL-23p19 7B7 heavy chain variable domain is shown in SEQ IDNO:7, and the light chain variable domain is shown in SEQ ID NO:9).

EXAMPLE 3 Generation of Anti-Human IL-23/IL-17A/F Bispecific Antibodies

Construction and Expression of Mammalian Anti-Human IL-23/IL-17A/FBispecific Molecules

Partial or whole genes were synthesized at GeneART, Inc. (GeneART, Inc.Burlingame, Calif., USA) or GenScript (GenScript, Piscataway, N.J., USA)and inserted into pTT5, an HEK293-6E transient expression vector (NCRBiotechnology Research Institute, Ottawa, ON, Canada) via restrictionenzyme cloning. MVC1059 (SEQ ID NO:62), and MVC1061 (SEQ ID NO:60) wereordered as complete constructs from GenScript (GenScript, Piscataway,N.J., USA). All constructs were expressed using the mod2610 (SEQ IDNO:30) signal sequence. The biAbFabL is a bispecific antibody whichcontains a whole antibody with a C-terminal Fab unit of the second armof the bispecific attached via a linker (e.g., 10 mer G₄S) and utilizesa common light chain (see FIG. 2). The taFab is a bispecific antibodywhich contains a whole antibody with an N-terminal Fab unit of thesecond arm of the bispecific attached via a linker, such as(Gly₄Ser₁)_(x), wherein x is 1, 2 or 3, and the linker of SEQ ID NO:12.As with the heavy chain portion, there are two light chains for each armof the bispecific attached via a linker, such as (Gly₄Ser₁)_(x), whereinx is 1, 2 or 3, and the linker of SEQ ID NO:12 (see FIG. 3). TheHeterodimeric Fc is a bispecific antibody that resembles a traditionalantibody, however, contains two different heavy chains which associatethrough an electrostatic complementarity association in the C_(H3)region. The Heterodimeric Fc utilizes a common light chain (see FIG. 4).Heavy chain and light chain constant regions include, IgG1.1 (SEQ IDNO:11, which may be encoded by SEQ ID NO:82), human kappa constantregion (SEQ ID NO:10, which may be encoded by SEQ ID NO:83), or IgG4.1 avariant of wild-type IgG4 that has a mutation in the hinge region, S228P(EU index numbering system) or S241P (Kabat numbering system). Changingthe serine at 241 (Kabat) to proline (found at that position in IgG1 andIgG2) in a mouse/human chimeric heavy chain leads to the production of ahomogeneous antibody and abolishes the heterogeneity. Further, thevariant IgG4 has significantly extended serum half-life and shows animproved tissue distribution compared to the original chimeric IgG4.Angal et al., Molecular Immunology, 30(1):105-108 (1993); Schuurman etal., Molecular Immunology, 38:1-8 (2001); Lewis et al., MolecularImmunology, 46:3488-3494 (2009).

Transformation of electrocompetent E. coli host cells (DH10B) wasperformed using 1 μl of the yeast DNA preparation and 20 μl of E. colicells. The cells were electropulsed at 2.0 kV, 25 μF, and 400 ohms.Following electroporation, 600 μl SOC (2% BACTO® Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) was added and the cells wereplated in 50 μl and 550 μl aliquots on two LB AMP plates (LB broth(Lennox), 1.8% BACTO® Agar (Difco), 100 mg/L Ampicillin).

Five colonies from each construct were subjected to sequence analysis.One clone containing the correct sequence was selected. DNA sequencingwas performed using ABI PRISM® BigDye Terminator v3.1 Cycle SequencingKit (Applied Biosystems, Foster City, Calif.). Sequencing reactions werepurified using Edge BioSystems Preforma Centriflex Gel FiltrationCartridges (Gaithersburg, Md.) and run on an Applied Biosystems 3730 DNAAnalyzer (Applied Biosystems, Foster City, Calif.). Resultant sequencedata was assembled and edited using SEQUENCHER® v4.6 software (GeneCodesCorporation, Ann Arbor, Mich.). One clone containing the correctsequence was selected and large-scale plasmid DNA was isolated using acommercially available kit (QIAGEN® Plasmid Mega Kit, Qiagen, Valencia,Calif.) according to manufacturer's instructions.

The HEK293-6E suspension cells were transfected with expressionconstructs using polyethylenimine reagent and cultivated in F17 medium(Invitrogen, Grand Island, N.Y., USA) with the addition of 5 mML-glutamine and 25 μg/mL G418. After 24 hours, 1/40th volume of 20%Tryptone NI (Organotechnie SAS, La Courneuve, FR) was added. Atapproximately 120 hours post transfection, conditioned media washarvested and passed through a 0.2 μm filter. Protein was purified fromthe filtered conditioned media using a combination of Mab Select SuReAffinity Chromatography (GE Healthcare, Piscataway, N.J., USA) andSUPERDEX® 200 Size Exclusion Chromatography (GE Healthcare, Piscataway,N.J., USA). Content was estimated by absorbance at UV-A280 nm andquality evaluated by analytical size exclusion high performance liquidchromatography, SDS PAGE, and western blot.

Anti-Human IL-23/IL-17A/F Bispecific Antibody Composition

A whole antibody and its modular components is depicted in FIG. 1. ThebiAbFabL format is depicted in FIG. 2. The taFab format is depicted inFIG. 3. The heterodimeric Fc format is depicted in FIG. 4. The VCVFcformat is depicted in FIG. 5. The VCDFc format is depicted in FIG. 6.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;NIH/3T3/KZ170 NF-κB Luciferase Reporter Assay to Measure Human IL-17A,IL-17A/F, and IL-17F Activity by NF-κB Induction

The material and methods for this assay are described in Example 1hereinabove.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Baf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay to MeasureHuman IL-23 Activity by Phospho-STAT3 Induction

A murine bone marrow derived cell line (Baf3) was stably transfectedwith human IL-23Rα and human IL-12Rβ1 and cloned.Baf3/huIL-23Rα/huIL-12Rβ1 clone 6 cells were washed three times withassay media (RPMI 1640 plus 10% fetal bovine serum, 2 mM L-Glutamine, 1mM Sodium Pyruvate (HyClone Laboratories, South Logan, Utah), and 2 μMβ-Mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.)) before being platedout at 50,000 cells/well in 96-well, round-bottom tissue culture plates.Serial dilutions of recombinant human IL-23 (ZymoGenetics, ABristol-Myers Squibb Company, Seattle Wash., USA) were made up in assaymedia and added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 15 minutes. Additionally the assay wasalso used to measure neutralization of IL-23 activity. A half maximalconcentration (EC₅₀, effective concentration at 50 percent) of IL-23 wascombined with serial dilutions of anti-human IL-23/IL-17A/F antibodiesdescribed herein and incubated together at 37° C., 5% CO₂ for 15 minutesin assay media prior to addition to cells. Following pre-incubation,treatments were added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 15 minutes. Following incubation, cellswere washed with ice-cold wash buffer and put on ice to stop thereaction according to manufacturer's instructions (BIO-PLEX® Cell LysisKit, Bio-Rad Laboratories, Hercules, Calif.). Cells were then spun downat 2000 rpm at 4° C. for 5 minutes prior to dumping the media. FiftyμL/well lysis buffer was added to each well; lysates were pipetted upand down five times while on ice, then agitated on a plate shaker for 20minutes at 300 rpm and 4° C. Plates were centrifuged at 3200 rpm at 4°C. for 20 minutes. Supernatants were collected and transferred to a newmicro titer plate for storage at −80° C.

Capture beads (BIO-PLEX® Phospho-STAT3 Assay, Bio-Rad Laboratories) werecombined with 50 μL of 1:1 diluted lysates and added to a 96-well filterplate according to manufacturer's instructions (BIO-PLEX® PhosphoproteinDetection Kit, Bio-Rad Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 μL/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed threetimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 μL/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX® 100, Bio-RadLaboratories) according to the manufacturer's instructions. Data wasanalyzed using analytical software (BIO-PLEX® Manager 4.1, Bio-RadLaboratories). Increases in the level of the phosphorylated STAT3transcription factor present in the lysates were indicative of an IL-23receptor-ligand interaction. Decreases in the level of thephosphorylated STAT3 transcription factor present in the lysates wereindicative of neutralization of the IL-23 receptor-ligand interaction.IC₅₀ (inhibitory concentration at 50 percent) values were calculatedusing GraphPad Prism 4 software (GraphPad Software, Inc., San DiegoCalif.) for each anti-human IL-23/IL-17A/F antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibody Bioassay Activity;NIH/3T3/KZ170 NF-κB Luciferase Reporter Assay andBaf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay Results

Human IL-17A, IL-17A/F and IL-17F induce activation of the NF-κBluciferase reporter in a dose dependent manner with an EC₅₀concentration determined to be 0.33 nM for IL-17A, 1 nM for IL-17A/F and1 nM for IL-17F and IL-23 induces STAT3 phosphorylation in a dosedependent manner with an EC₅₀ concentration determined to be 0.02 nM.The IC₅₀ data for the anti-human IL-23/IL-17A/F bispecific antibodies isshown below in Table 8.

Anti-Human IL-23/17A/F Bispecific Antibody Table

TABLE 8 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339-134MVC978 MVC717 1.3 0.27 0.24 Not Done mAb IgG1.1 SEQ ID SEQ ID NO: 64 NO:66 IL23.6 (7B7) MVC1003 MVC1002 Not Done Not Done Not Done 0.014 mAbIgG1.1 SEQ ID SEQ ID NO: 68 NO: 17* 23/17bAb1 MVC1006 MVC1002 0.064 0.760.96 0.015 IgG1.1 SEQ ID SEQ ID NO: 28* NO: 17* 23/17bAb2 MVC1007MVC1002 0.052 0.43 0.44 0.041 IgG1.1 SEQ ID SEQ ID NO: 18* NO: 17*23/17bAb3 MVC1036 MVC1002 0.022 0.20 0.23 0.012 IgG4.1 SEQ ID SEQ ID NO:74 NO: 17* 23/17bAb4 MVC1037 MVC1002 0.035 0.18 0.87 0.048 IgG4.1 SEQ IDSEQ ID NO: 29* NO: 17* 23/17taFab1 MVC1008 MVC 1009 1.5 3.9 2.3 0.018IgG1.1 SEQ ID SEQ ID NO: 76 NO: 78 23/17hetero1 MVC1059 MVC1002 0.340.78 0.33 0.060 IgG1.1 SEQ ID SEQ ID NO: 62 NO: 17* MVC1060 SEQ ID NO:64 23/17hetero2 MVC1061 MVC1002 0.71 2.33 0.96 0.055 IgG1.1 SEQ ID SEQID NO: 60 NO: 17* MVC1062 SEQ ID NO: 80 *The amino acid sequence of SEQID NO: 17 may be encoded by the sequence of SEQ ID NO: 70; the aminoacid sequence of SEQ ID NO: 28 may be encoded by the sequence of SEQ IDNO: 71; the amino acid sequence of SEQ ID NO: 18 may be encoded by thesequence of SEQ ID NO: 72; the amino acid sequence of SEQ ID NO: 29 maybe encoded by the sequence of SEQ ID NO: 75.Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human SAEC Assay to Measure Human IL-17A, IL-17AF, and IL-17FActivity by G-CSF Induction

Primary human small airway epithelial cells (SAEC) were seeded at 8,000cells/well in Small Airway Epithelial Growth Medium (SAGM) (cells andmedia: Lonza, Walkersville, Md.) in 96-well flat bottom tissue cultureplates (Corning Incorporated, Corning, N.Y.) and incubated overnight at37° C., 5% CO₂. The following day serial dilutions of human IL-17A,IL-17A/F, or IL-17F (ZymoGenetics, A Bristol-Myers Squibb Company,Seattle, Wash., USA) were made up in SAGM media and added to the platescontaining the cells and incubated together at 37° C., 5% CO₂ for 24hours. Additionally the assay was used to measure neutralization ofIL-17A, IL-17A/F and IL-17F activity. A half maximal concentration(EC₅₀, effective concentration at 50 percent) of IL-17A, IL-17A/F orIL-17F was combined with serial dilutions of anti-human IL-23/IL-17A/Fbispecific antibodies described herein in SAGM media and added to theplates containing the cells and incubated together at 37° C., 5% CO₂ for24 hours. After incubation the supernatants were spun down, collectedand frozen at −80° C. until ready to process. Human G-CSF protein levelsin the supernatants were measured using a commercial bead based humanG-CSF cytokine ELISA according to manufactures instructions(Procarta/Affymetrix, Santa Clara, Calif.). Increases in human G-CSFlevels in the supernatant were indicative of a human IL-17A, IL-17A/F,IL-17F receptor-ligand interaction. Decreases in human G-CSF levels inthe supernatant were indicative of neutralization of the human IL-17A,IL-17A/F, IL-17F receptor-ligand interaction. IC₅₀ (inhibitoryconcentration at 50 percent) values were calculated using GraphPad Prism4 software (GraphPad Software, Inc., San Diego Calif.) for eachanti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human SAEC Assay Results

Human IL-17A, IL-17A/F and IL-17F induce human G-CSF production in adose dependent manner with an EC₅₀ concentration determined to be 0.03nM for IL-17A, 3 nM for IL-17A/F and 3 nM for IL-17F. Bispecificantibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17),23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 andSEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). The humanizedanti-human IL-17A/F antibody 339-134 mAb (SEQ ID NO:65 and SEQ ID NO:67)was also tested. The IC₅₀ data for the anti-human IL-23/IL-17A/Fbispecific antibodies is shown below in Table 9. This data indicatesthat the anti-IL-23/IL-17A/F bispecific antibodies inhibit human IL-17A,IL-17A/F, IL-17F mediated IL-6 production were equally potent.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human Fibroblast Assay to Measure Human IL-17A, IL-17A/F, andIL-17F Activity by IL-6 Induction

A primary human fibroblast cell line (HFFF2, Cat #86031405, HealthProtection Agency Culture Collections, Porton Down Salisbury, UK) wasseeded at 5,000 cells/well in assay media (DMEM plus 10% FBS and 2 mML-glutamine (HyClone Laboratories, South Logan, Utah)) in 96-well flatbottom plates (Corning Incorporated, Corning, N.Y.) and incubatedovernight at 37° C., 5% CO₂. The following day serial dilutions ofrecombinant human IL-17A, IL-17AF, or IL-17F (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash. 98117) were made up inassay media and added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 24 hours. Additionally the assay was usedto measure neutralization of human IL-17A, IL-17A/F and IL-17F activity.A half maximal concentration (EC₅₀, effective concentration at 50percent) of human IL-17A, IL-17A/F or IL-17F was combined with serialdilutions of anti-human IL-23/IL-17A/F antibodies described herein inassay media and added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 24 hours. After incubation thesupernatants were spun down, collected and frozen at −80° C. until readyto process. Human IL-6 protein levels in the supernatants were measuredusing a commercial bead based human IL-6 cytokine ELISA according tomanufactures instructions (Bio-Rad Laboratories, Hercules, Calif.).Increases in human IL-6 levels in the supernatant were indicative of ahuman IL-17A, IL-17A/F, IL-17F receptor-ligand interaction. Decreases inhuman IL-6 levels in the supernatant were indicative of neutralizationof the human IL-17A, IL-17A/F, IL-17F receptor-ligand interaction. IC₅₀(inhibitory concentration at 50 percent) values were calculated usingGraphPad Prism 4 software (GraphPad Software, Inc., San Diego Calif.)for each anti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human Fibroblast Assay Results

Human IL-17A, IL-17A/F and IL-17F induce human IL-6 production in a dosedependent manner with an EC₅₀ concentration determined to be 0.08 nM forIL-17A, 25 nM for IL-17AF and 25 nM for IL-17F. Bispecific antibodiestested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17), 23/17bAb2 (SEQID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 and SEQ ID NO:17),23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). Humanized anti-human IL-17A/Fantibody 339-134 mAb (SEQ ID NO:64 and SEQ ID NO:66) was also tested.The IC₅₀ data for the anti-human IL-23/IL-17A/F bispecific antibodies isshown below in Table 9. These data indicate that the anti-humanIL-23/IL-17A/F bispecific antibodies inhibit human IL-17A, IL-17A/F,IL-17F mediated IL-6 production were equally potent.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Murine Splenocyte Assay to Measure Human IL-23 Activity by Murine IL-17Aand IL-17F Induction

A single cell suspension of murine splenocytes was prepared from wholespleens harvested from BALB/c mice. After red blood cell lysis with ACKbuffer (0.010 M KHCO₃, 0.0001 M EDTA, 0.150 M NH₄Cl, pH 7.2) splenocyteswere washed and resuspended in assay media (RPMI 1640 plus 10% FBS,non-essential amino acids, 1 mM Sodium Pyruvate, 2 mM L-glutamine, 10 mMHEPES, 100 units/mL Pen/Strep (HyClone Laboratories, South Logan, Utah),50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.), and 50 ng/mlhuman IL-2 (R&D Systems, Minneapolis, Minn.)). Splenocytes were seededat 500,000 cells per well in 96-well round bottom plates. Serialdilutions of recombinant human IL-23 (BDC 50220AN087 heterodimermaterial) were made up in assay media and added to the plates containingthe cells and incubated together at 37° C., 5% CO₂ for 24 hours.Additionally the assay was also used to measure neutralization of humanIL-23 activity. A half maximal concentration (EC₅₀, effectiveconcentration at 50 percent) of human IL-23 was combined with serialdilutions of anti-human IL-23/IL-17A/F bispecific antibodies describedherein and incubated together at 37° C., 5% CO₂ for 15 minutes in assaymedia prior to addition to cells. Following pre-incubation, treatmentswere added to the plates containing the cells and incubated together at37° C., 5% CO₂ for 24 hours. After incubation the supernatants were spundown, collected and frozen at −80° C. until ready to process. Theprotein levels of murine IL-17A and IL-17F in the supernatants weremeasured using commercial plate based murine IL-17A and IL-17F ELISA'saccording to manufacturer's instructions (eBiosciences, San Diego,Calif.). Increases in murine IL-17A and IL-17F levels in the supernatantwere indicative of an IL-23 receptor-ligand interaction. Decreases inmurine IL-17A and IL-17F levels in the supernatant were indicative ofneutralization of the IL-23 receptor-ligand interaction. IC₅₀(inhibitory concentration at 50 percent) values were calculated usingGraphPad Prism 4 software (GraphPad Software, Inc., San Diego Calif.)for each anti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Murine Splenocyte Assay Results

Human IL-23 induced murine IL-17A and IL-17F in a dose dependent mannerwith an EC₅₀ concentration determined to be 0.01 nM. Bispecificantibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17),23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 andSEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). The anti-humanIL-23.6 (7B7) mAb (SEQ ID NO:68 and SEQ ID NO:17) was also tested. TheIC₅₀ data for the anti-human IL-23/IL-17A/F bispecific antibodies isshown below in Table 9. This data indicates that the anti-humanIL-23/IL-17A/F bispecific antibodies inhibit human IL-23 induced murineIL-17A and IL-17F production.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human T Cell Phospho-STAT3 Assay to Measure Human IL-23 Activityby Phospho-STAT3 Induction

Leukopheresis PBMC: Normal human donors (ZymoGenetics' normal donorpool) were selected at random and were voluntarily apheresed at theFHCRC (Seattle, Wash.). The leukopheresis PBMC were delivered toZymoGenetics in a sterile blood-collection bag. The cells were pouredinto a sterile 500 mL plastic bottle, diluted to 400 mL with roomtemperature PBS plus 1 mM EDTA (HyClone Laboratories, South Logan, Utah)and transferred to 250 mL conical tubes. The 250 mL tubes werecentrifuged at 1500 rpm for 10 minutes to pellet the cells. The cellsupernatant was then removed and discarded. The cell pellets were thencombined and suspended in 400 mL PBS plus 1 mM EDTA. The cell suspension(25 mL/tube) was overlaid onto FICOLL® (20 mL/tube) in 50 mL conicaltubes (total of 16 tubes). The tubes were centrifuged at 2000 rpm for 20minutes at room temperature. The interface layer (“buffy coat”)containing the white blood cells and residual platelets was collected,pooled and washed repeatedly with PBS plus 1 mM EDTA until the majorityof the platelets had been removed. The white blood cells were thensuspended in 100 mL of ice-cold Cryopreservation medium (70% RPMI 1640,20% FCS, 10% DMSO (HyClone Laboratories)) and distributed into sterilecryovials (1 mL cells/vial). The cryovials placed in a −80° C. freezerfor 24 hours before transfer to a liquid-nitrogen freezer. The whiteblood-cell yield from a typical apheresis is 0.5-1.0×10¹⁰ cells.Apheresis cells processed in this manner contain T cells, B cells, NKcells, monocytes and dendritic cells.

Preparation of Activated T Cells:

T cells must be activated in order to express the IL-12 receptor and beable to respond to IL-12 and IL-23. Cryopreserved leukopheresis PBMCwere thawed, transferred to a sterile 50 mL conical tube, and washedwith 50 mL of warm assay media (RPMI 1640 plus 10% FBS (HyCloneLaboratories)) and incubated in a 37° C. water bath for 1 hour to allowthe cells to recover.

The cells were then centrifuged and the cell-supernatant discarded. Thecell pellet was resuspended in assay media and distributed into sterile162 cm² tissue culture flasks at 2×10⁷ cells per flask in 90 mL assaymedia containing 5 μg/mL PHA-M (Roche, Basel, Switzerland). The cellswere then cultured at 37° C. in a humidified incubator for a total of 5days. The cells were “rested” by harvesting on the afternoon of day 4,replacing the culture medium with fresh assay media without PHA andreturning to the incubator for the remainder of the 5 day cultureperiod.

Phospho-STAT3 Assay:

Activated human T cells were harvested on day 5 of culture andresuspended in fresh assay media and were plated out at 2×10⁵ cells/wellin U-bottom 96-well plates. Serial dilutions of recombinant human IL-23(BDC 50220AN087 heterodimer material) were made up in assay media andadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 15 minutes. Additionally the assay was also used tomeasure neutralization of IL-23 activity. A half maximal concentration(EC₅₀, effective concentration at 50 percent) of IL-23 was combined withserial dilutions of anti-human IL-23/IL-17AF antibodies described hereinand incubated together at 37° C., 5% CO₂ for 15 minutes in assay mediaprior to addition to cells. Following pre-incubation, treatments wereadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 15 minutes. Following incubation, cells were washed withice-cold wash buffer and put on ice to stop the reaction according tomanufacturer's instructions (BIO-PLEX® Cell Lysis Kit, Bio-RadLaboratories, Hercules, Calif.). Cells were then spun down at 2000 rpmat 4° C. for 5 minutes prior to dumping the media. Fifty 4/well lysisbuffer was added to each well; lysates were pipetted up and down fivetimes while on ice, then agitated on a plate shaker for 20 minutes at300 rpm and 4° C. Plates were centrifuged at 3200 rpm at 4° C. for 20minutes. Supernatants were collected and transferred to a new microtiter plate for storage at −80° C.

Capture beads (BIO-PLEX® Phospho-STAT3 Assay, Bio-Rad Laboratories) werecombined with 50 μL of 1:1 diluted lysates and added to a 96-well filterplate according to manufacturer's instructions (BIO-PLEX® PhosphoproteinDetection Kit, Bio-Rad Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 μL/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed threetimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 μL/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX® 100, Bio-RadLaboratories) according to the manufacturer's instructions. Data wasanalyzed using analytical software (BIO-PLEX® Manager 4.1, Bio-RadLaboratories). Increases in the level of the phosphorylated STAT3transcription factor present in the lysates were indicative of an IL-23receptor-ligand interaction. Decreases in the level of thephosphorylated STAT3 transcription factor present in the lysates wereindicative of neutralization of the IL-23 receptor-ligand interaction.IC₅₀ (inhibitory concentration at 50 percent) values were calculatedusing GraphPad Prism 4 software (GraphPad Software, Inc., San DiegoCalif.) for each anti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human T Cell Phospho-STAT3 Assay Results

Human IL-23 induces STAT3 phosphorylation in a dose dependent mannerwith an EC₅₀ concentration determined to be 0.02 nM. Bispecificantibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17),23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 andSEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). The anti-humanIL-23.6 (7B7) mAb (SEQ ID NO:68 and SEQ ID NO:17) was also tested. TheIC₅₀ data for the anti-human IL-23/IL-17A/F antibodies is shown below inTable 9.

Anti-Human IL-23/IL-17A/F Bispecific Antibody Bioassay Activity; PrimaryHuman SAEC Assay, Primary Human Fibroblast Assay, Murine SplenocyteAssay and Primary Human T Cell Phospho-STAT3 Assay Results

TABLE 9 23/17bAb1 23/17bAb2 23/17bAb3 23/17bAb4 339-134 IL23.6(7B7)IgG1.1 IgG1.1 IgG4.1 IgG4.1 mAbIgG1.1 mAbIgG1.1 SEQ ID NO: 28 SEQ ID NO:18 SEQ ID NO: 74 SEQ ID NO: 29 SEQ ID NO: 64 SEQ ID NO: 68 Profile SEQID NO: 17 SEQ ID NO: 17 SEQ ID NO: 17 SEQ ID NO: 17 SEQ ID NO: 66 SEQ IDNO: 17 Cellular Potency IL-17A <0.5 pM <0.5 pM <0.5 pM ≦0.5 pM 0.5 nMNot Done Hu. primary EC₅₀ = epithelial cells 0.03 nM (SAEC) IL-17AF 1.4nM 1.3 nM 0.5 nM 1.4 nM 1.3 nM Not Done IC₅₀ EC₅₀ = 3 nM IL-17F 0.8 nM1.6 nM 1.0 nM 1.3 nM 1.1 nM Not Done EC₅₀ = 3 nM Cellular Potency IL-17A0.07 nM 0.07 nM 0.03 nM 0.1 nM 0.9 nM Not Done Hu. primary EC₅₀ =fibroblast cells 0.08 nM (HFFF) IL-17AF 17 nM 12 nM 9.4 nM 9.1 nM 13 nMNot Done IC₅₀ EC₅₀ = 25 nM IL-17F 19 nM 15 nM 10 nM 12 nM 15 nM Not DoneEC₅₀ = 25 nM Cellular potency IL-23 0.1 nM 0.06 nM 0.1 nM 0.08 nM NotDone 0.09 nM Murine splenocyte EC₅₀ = assay IC₅₀ 0.01 nM Cellularpotency IL-23 0.04 nM 0.05 nM 0.04 nM 0.1 nM Not Done 0.04 nM Primary Tcell EC₅₀ = assay IC₅₀ 0.02 nM

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Co-Binding Activity;Primary Human Fibroblast Assay to measure the inhibition of humanIL-17A, IL-7A/F, or IL-F while simultaneously bound to human IL-23. ThePrimary Human T Cell Phospho-STAT3 Assay to measure the inhibition ofhuman IL-23 while simultaneously bound to human IL-17A, IL-7A/F, orIL-17F.

The primary human fibroblast assay was run in the presence of excessamounts of IL-23 at 30 nM. The primary human T cell phospho-STAT3 assaywas run in the presence of excess amounts of IL-17A, IL-17A/F, IL-17F at30 nM.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Co-BindingResults

Bispecific antibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ IDNO:17), 23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ IDNO:74 and SEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17), and23/17taFab1 (SEQ ID NO:76 and SEQ ID NO:78). The anti-humanIL-23/IL-17A/F bispecific antibodies when examined in the presence ofhuman IL-23 did not interfere with human IL-17A, IL-17A/F, IL-17Finhibition. The anti-human IL-23/IL-17A/F bispecific antibodies whenexamined in the presence of human IL-17A, IL-17A/F, IL-17F did notinterfere with human IL-23 inhibition.

Measurement of Binding Affinities of Anti-Human IL-23/IL-17A/FBispecific Antibodies to Human IL-17A, IL-17A/F, IL-17F, and Human IL-23Via Surface Plasmon Resonance (Biacore)

Anti-human IL-23/IL-17A/F bispecific antibodies were evaluated for theirbinding affinity to human IL-17A, human IL-17A/F, human IL-17F, andhuman IL-23 using surface plasmon resonance.

Kinetic rate constants and equilibrium dissociation constants weremeasured for the interaction of the anti-human IL-23/IL-17A/F bispecificantibodies with human IL-17A, IL-17A/F, IL-17F, and human IL-23 viasurface plasmon resonance. The association rate constant (k_(a)(M⁻¹s⁻¹)) is a value that reflects the rate of the antigen-antibodycomplex formation. The dissociation rate constant (k_(d) (s⁻¹)) is avalue that reflects the stability of this complex. By dividing thedissociation rate constant by the association rate constant(k_(d)/k_(a)) the equilibrium dissociation constant (K_(D) (M)) isobtained. This value describes the binding affinity of the interaction.Antibodies with similar K_(D) can have widely variable association anddissociation rate constants. Consequently, measuring both the k_(a) andk_(d) of antibodies helps to more uniquely describe the affinity of theantibody-antigen interaction.

Binding kinetics and affinity studies were performed on a BIACORE® T100system (GE Healthcare, Piscataway, N.J.). Methods for the BIACORE® T100were programmed using BIACORE® T100 Control Software, v 2.0. For theseexperiments, the monoclonal and bispecific antibodies were captured ontoa CM4 sensor chip via goat anti-human IgG Fc-gamma antibody (JacksonImmunoResearch, West Grove, Pa.). Binding experiments with the humanIL-17 molecules were performed at 25° C. in a buffer of 10 mM HEPES, 150mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GE Healthcare), 1 mg/mL bovineserum albumin, pH 7.4. Binding experiments with the IL-23/IL-12Bheterodimer were performed at 25° C. in a buffer of 10 mM HEPES, 500 mMNaCl, 3 mM EDTA, 0.05% Surfactant P20 (Biacore), 1 mg/mL bovine serumalbumin, pH 7.4.

The capture antibody, goat anti-human IgG Fc-gamma, was diluted toconcentration of 20 μg/mL in 10 mM sodium acetate pH 5.0, and thencovalently immobilized to all four flow cells of a CM4 sensor chip usingamine coupling chemistry (EDC:NHS). After immobilization of theantibody, the remaining active sites on the flow cell were blocked with1 M ethanolamine. A capture antibody density of approximately 5000 RUwas obtained. The anti-human IL-23/IL-17A/F antibodies were capturedonto flow cell 2, 3, or 4 of the CM4 chip at a density ranging from60-150 RU. Capture of the test antibodies to the immobilized surface wasperformed at a flow rate of 10 μL/min. The BIACORE® instrument measuresthe mass of protein bound to the sensor chip surface, and thus, captureof the test antibody was verified for each cycle. Serial dilutions ofhuman recombinant IL-17A, IL-17A/F, or IL-17F (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA) were prepared from100 nM-0.032 nM (1:5 serial dilutions), while serial dilutions of humanrecombinant IL-23 (ZymoGenetics, A Bristol-Myers Squibb Company,Seattle, Wash., USA) were prepared from 200 nM-0.064 nM (1:5 serialdilutions). The serial dilutions were injected over the surface andallowed to specifically bind to the test antibody captured on the sensorchip. Duplicate injections of each antigen concentration were performedwith an association time of 7 minutes and dissociation time of 15minutes. Kinetic binding studies were performed with a flow rate of 50μL/min. In between cycles, the flow cell was washed with 20 mMhydrochloric acid to regenerate the surface. This wash step removed boththe captured test antibody and any bound antigen from the immobilizedantibody surface. The test antibody was subsequently captured again inthe next cycle.

Data was compiled using the BIACORE® T100 Evaluation software (version2.0). Data was processed by subtracting reference flow cell and blankinjections. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Duplicate injection curves were checked forreproducibility. Based on the binding of the bivalent IL-17 molecules toa bivalent antibody, the bivalent analyte binding interaction model wasdetermined to be appropriate for interactions with the IL-17 molecules.Based on the binding of the IL-23/IL-12B heterodimer to a bivalentantibody, the 1:1 binding interaction model was determined to beappropriate for interactions with the IL-23 molecule. The referencesubtracted binding curves were globally fit to the appropriate bindingmodel with a multiple Rmax and with the RI set to zero. The data fitwell to the binding models with good agreement between the experimentaland theoretical binding curves. The chi² and standard errors associatedthe fits were low. There was no trending in the residuals.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Biacore Activity

The results of the binding experiments with human IL-17A, IL-17A/F, andIL-17F are shown in Tables 10, 11, and 12, respectively. The results ofthe binding experiments with the human IL-23/IL-12B heterodimer areshown in Table 13.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL-17A

TABLE 10 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)23/17bAb1 2.E+05 6.E−05 3.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 5.E+05 4.E−04 8.E−10 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 4.E+05 5.E−05 1.E−10 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 5.E+05 3.E−04 6.E−10 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 3.E+05 2.E−03 7.E−9 IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL-17A/F

TABLE 11 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)23/17bAb1 2.E+05 9.E−05 4.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 4.E+05 7.E−04 2.E−9 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 2.E+05 2.E−04 1.E−9 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 3.E+05 1.E−03 3.E−9 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 1.E+05 5.E−04 5.E−9 IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL-17F

TABLE 12 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)23/17bAb1 8.E+05 3.E−04 4.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 3.E+06 7.E−04 2.E−10 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 6.E+05 2.E−04 3.E−10 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 2.E+06 7.E−04 4.E−10 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 3.E+05 7.E−04 2.E−9 IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL23/IL-12B

TABLE 13 Antibody or k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹)K_(D1) (M) 7B7Mab 3.E+05 2.E−04 7.E−10 SEQ ID NO: 68 SEQ ID NO: 1723/17bAb1 4.E+05 2.E−04 5.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 2.E+05 8.E−05 4.E−10 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 4.E+05 2.E−04 5.E−10 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 7.E+04 7.E−05 1.E−9 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 3.E+05 2.E−04 7.E−10 IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Simultaneous Co-Binding of IL-17A/F and IL-23 to the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore)

Anti-Human IL-23/IL-17A/F bispecific antibodies were evaluated viasurface plasmon resonance for ability to simultaneously co-bind bothIL-23 and IL-17A/F.

For co-binding experiments in the first orientation, the human IL-17molecules were covalently immobilized to flow cells 2-4 of a CM5 sensorchip using amine coupling chemistry (EDC:NHS). After immobilization, theremaining active sites on the flow cells were blocked with 1 Methanolamine. Human IL-17A, IL-17A/F, and IL-17F (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA) were immobilized ontoflow cells 2, 3, or 4 respectively. The immobilization levels of thesemolecules ranged from 4500-5200 RU. Flow cell 1 was used as thereference surface. The bispecific antibodies were subsequently dilutedto either 25 or 50 μg/mL, flowed over the surface, and captured ontoflow cells 2-4 of the sensor chip. Following capture of the bispecificantibody, the IL-23/IL-12B heterodimer (ZymoGenetics, A Bristol-MyersSquibb Company, Seattle, Wash., USA) was diluted to 500 nM and flowedover the surface to demonstrate co-binding. Binding studies wereperformed with a flow rate of 10 μL/min, an association time of 10minutes, and a dissociation time of 5 minutes.

For co-binding experiments in the second orientation, a mouse anti-humanIL-12 (p40/p70) monoclonal antibody (BD Pharmingen, San Jose, Calif.)was covalently immobilized onto flow cells 1-4 of a CM5 sensor chipusing amine coupling chemistry (EDC:NHS). After immobilization, theremaining active sites on the flow cells were blocked with 1 Methanolamine. The human IL-23/IL-12B heterodimer (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA) was diluted to 500 nMand captured onto flow cells 1-4 via the IL-12B subunit. The capturelevel of the IL-23/IL-12B was approximately 4000 RU. The bispecificantibodies were subsequently diluted to either 25 or 50 μg/mL, flowedover the surface, and captured via the human IL-23 subunit onto flowcells 2-4 of the sensor chip. Flow cell 1 was used as the referencesurface. Following capture of the bispecific antibody, human IL-17A,IL-17A/F, and IL-17F (ZymoGenetics, A Bristol-Myers Squibb Company,Seattle, Wash., USA) were diluted to 500 nM and flowed over the surfaceto demonstrate co-binding. Binding studies were performed with a flowrate of 10 μL/min, an association time of 10 minutes, and a dissociationtime of 5 minutes.

All binding experiments were performed at 25° C. in a buffer of 10 mMHEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GE Healthcare), 1mg/mL bovine serum albumin, pH 7.4. Between cycles, the flow cell waswashed with 20 mM hydrochloric acid to regenerate the surface. This washstep removed both the captured test antibody and any bound antigen fromthe chip surface. Data was compiled using BIACORE® T100 Evaluationsoftware (version 2.0). Data was processed by subtracting reference flowcell and blank injections. Baseline stability was assessed to ensurethat the regeneration step provided a consistent binding surfacethroughout the sequence of injections.

Simultaneous Co-Binding of IL-17A/F and IL-23 to the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore) Results

Bispecific antibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ IDNO:17), 23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ IDNO:74 and SEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17), and23/17taFab1 (SEQ ID NO:76 and SEQ ID NO:78). All bispecific antibodieswere able to simultaneously co-bind both human IL-23 and human IL-17A/F,demonstrating that both arms of the bispecific antibodies werefunctional.

Demonstration of IL-17A/F Specific Binding of the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore)

Anti-Human IL-23/IL-17A/F bispecific antibodies were evaluated viasurface plasmon resonance for lack of cross reactivity to human IL-17B,human IL-17C, human IL-17D, and human IL-17E (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA).

Binding studies were performed on a BIACORE® T100 (GE Healthcare,Piscataway, N.J.). Methods were programmed using BIACORE® T100 ControlSoftware, v 2.0. Goat anti-human IgG Fc-gamma specific antibody (JacksonImmunoResearch, West Grove, Pa.) was covalently immobilized to flowcells 1-3 of a CM4 sensor chip using amine coupling chemistry (EDC:NHS).The purified bispecific antibodies were subsequently captured ontoeither flow cell 2 or flow cell 3 of the sensor chip at a density ofapproximately 150 RU. Flow cell 1 was used as the reference surface.

Human IL-17B, IL-17C, IL-17D, and IL-17E (ZymoGenetics, A Bristol-MyersSquibb Company, Seattle, Wash., USA) were injected over the capturedantibody surface (flow cell 2) and the reference flow cell (flow cell 1)at concentrations of 500, 100, 20, and 4 nM. As a positive control forthis set of experiments, human IL-23 (ZymoGenetics, A Bristol-MyersSquibb Company, Seattle, Wash., USA) was injected at concentrations of100, 20, 4 and 0.8 nM. Binding studies were performed with a flow rateof 50 μL/min, an association time of 5 minutes, and a dissociation timeof 5 minutes. All binding experiments were performed at 25° C. in abuffer of 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GEHealthcare), 1 mg/mL bovine serum albumin, pH 7.4. Between cycles, theflow cell was washed with 20 mM hydrochloric acid to regenerate thesurface. This wash step removed both the captured test antibody and anybound antigen from the chip surface. Data was compiled using BIACORE®T100 Evaluation software (version 2.0). Data was processed bysubtracting reference flow cell and blank injections. Baseline stabilitywas assessed to ensure that the regeneration step provided a consistentbinding surface throughout the sequence of injections.

Demonstration of IL-17A/F Specific Binding of the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore) Results

No binding of human IL-17B, IL-17C, IL-17D, or IL17E to the bispecificantibodies was observed. Bispecific antibodies tested include 23/17bAb1(SEQ ID NO:28 and SEQ ID NO:17), 23/17bAb2 (SEQ ID NO:18 and SEQ IDNO:17), 23/17bAb3 (SEQ ID NO:74 and SEQ ID NO:17), 23/17bAb4 (SEQ IDNO:29 and SEQ ID NO:17). In contrast, the IL-23 positive controldemonstrated a dose dependent binding that was consistent with theprevious studies.

EXAMPLE 4 Anti-Human IL-23/17A/F bAbs Prevent Human IL-17A, F andAF-Mediated Increases in Serum Concentrations of Murine KC(CXCL1) inMice

IL-17A, F and AF are able to induce the production of a number ofdownstream factors that in turn play a role in host defense, but alsocontribute to disease pathology, especially when produced at abnormallyhigh levels or under chronic conditions. One of these downstreammediators is CXCL1 (also known as GRO-α in human, or KC in mice), achemokine that has important neutrophil chemoattractant activity andplays a role in inflammation. The ability of anti-human IL23/17A/Fbispecific antibodies (bAbs) to reduce IL-17A, F and AF-mediatedincreases in GRO-α in mice was evaluated in order to show that the bAbswould be efficacious against IL-17-induced activities in an in vivosetting and thus that the bAbs would be useful in treating humandiseases in which IL-17A, F or AF play a role. However, because thesebAbs do not cross react with mouse IL-17A, F or AF, it was necessary todeliver human (h) IL-17A, F or AF to mice to induce the production ofGRO-α (or in the case of mice, induce the production of KC, which is themurine analogue of GRO-α) which could then be neutralized in thepresence of the anti-human IL23/17A/F bAbs.

For these experiments, female BALB/c mice (age 7-9 wk) were used. Attime 18 hours, the mice received an intra-peritoneal (i.p.) injection ofeither the vehicle (PBS) or a dose of one of the anti-human IL-23/17A/FbAbs as shown in Table 14 and 15, in the left-hand column. At time 0,they received a subcutaneous (s.c.) injection of one of the followingrecombinant human proteins: 0.175 mg/kg hIL-17A, 0.9 mg/kg hIL-17F or0.5 mg/kg hIL-17AF. Control mice received a s.c. injection of thevehicle (PBS) instead of one of the hIL-17 proteins. Two hours later,the mice were bled via the retro-orbital sinus under isoflurane gasanesthesia, serum was collected following centrifugation of the blood,and the serum was then stored at 80° C. until analyzed for serum KCconcentrations using a commercial ELISA as per the manufacturer'sinstructions (Quantikine Mouse CXCL1/KC Immunoassay, R&D Systems, Inc.,Minneapolis, Minn.).

As shown in Table 14 and 15, mice treated with the bAbs showed adose-dependent increase in the inhibition of hIL-17A, F or AF-inducedserum KC (CXCL1) concentrations indicating that the bAbs wereefficacious in reducing the activities mediated by these IL-17 ligands.CXCL1 is just one example of a biological readout in response to IL-17A,F or AF; there are numerous other important downstream readouts thatalso play a role in diseases in which IL-17A, F or AF play a role thatcould be used as endpoint measurement.

TABLE 14 Percent Inhibition of Human IL-17A or F-mediated Increases inSerum Concentrations of Murine KC by i.p. bAbs, Relative to theConcentrations of Vehicle-Treated Mice (n = 3-4 per Group) % Inhibition% Inhibition of of IL-17A-Mediated Serum IL-17F-Mediated Serum KC LevelsKC Levels Vehicle (PBS) 0 0  1 mg/kg bAb1 81 76  5 mg/kg bAb1 90 88 12mg/kg bAb1 100 97  1 mg/kg bAb2 91 68  5 mg/kg bAb2 93 83 12 mg/kg bAb284 90  1 mg/kg bAb3 78 95  5 mg/kg bAb3 94 89 12 mg/kg bAb3 93 90  1mg/kg bAb4 94 51  5 mg/kg bAb4 87 89 12 mg/kg bAb4 94 92

TABLE 15 Percent Inhibition of Human IL-17AF-mediated Increases in SerumConcentrations of Murine KC (pg/mL) by i.p. bAbs, Relative to theConcentrations of Vehicle-Treated Mice (n = 4 per Group) % Inhibition ofIL-17AF-Mediated Serum KC Levels Vehicle (PBS) 0 0.3 mg/kg bAb1 55  10mg/kg bAb1 100 0.3 mg/kg bAb2 40  10 mg/kg bAb2 90 0.3 mg/kg bAb3 70  10mg/kg bAb3 96 0.3 mg/kg bAb4 10  10 mg/kg bAb4 72

EXAMPLE 5 Anti-Human IL-23/17A/F bAbs Prevent Human IL-23-MediatedIncreases in Serum Concentrations of Mouse IL-17AF and F in Mice

IL-23 is able to induce the differentiation of Th17 cells which in turn,can lead to the production of IL-17A, IL-17F and IL-17AF. Thesecytokines are implicated in a number of diseases and therapeutics thatcan inhibit IL-23 and IL-17A, F and AF would be efficacious in thetreatment of these diseases. The ability of anti-human IL23/17A/Fbispecific antibodies (bAbs) to reduce IL-23-mediated increases inIL-17A, F and AF in mice was evaluated in order to show that the bAbswould be efficacious against IL-23-induced activities in an in vivosetting, and thus that the bAbs would be useful in treating humandiseases in which IL-23 and Th17 cells play a role. However, becausethese bAbs do not cross react with mouse IL-23 it was necessary todeliver human (h) IL-23 to mice to induce the production of mouse IL-17F and AF which could then be neutralized in the presence of theanti-human IL23/17A/F bAbs. Concentrations of mouse IL-17A were too lowto accurately measure in the mouse serum but the trends were expected tobe similar as compared to the trends observed for serum IL-17F and AF.

For these experiments, female C57BL/6 mice (age 7-9 wk) were used. At10:30 am on day 1, they each received 5 micrograms of mouse (m) IL-2 viaan intra-peritoneal (i.p.) injection. At 8:30 am on day 2, the micereceived an i.p. injection of either the vehicle (PBS) or a dose of oneof the anti-human IL-23/17A/F bAbs as shown in Table 16, in theleft-hand column. At 11 am on day 2 the mice each received 5 microgramsof mIL-2 and 10 micrograms of hIL-23, and at 5:20 pm on day 2, the micereceived 10 micrograms each of mIL-2 and hIL-23 via i.p. injections. At9:30 am on day 3, each of the mice received another 5 micrograms ofmIL-2 and 10 micrograms of hIL-23 by i.p. injection. At 4:30 pm on day3, the mice were bled via the retro-orbital sinus under isoflurane gasanesthesia, serum was collected following centrifugation of the blood,and the serum stored at −80° C. until analyzed for serum concentrationsof mouse IL-17F and AF using ELISAs and luminex assays that specificallymeasured these components.

As shown in Table 16, mice treated with the bAbs showed a dose-dependentincrease in the inhibition of hIL-23 induced serum concentrations ofmouse 17F or AF indicating that the bAbs were efficacious in reducingthe activities mediated by hIL-23.

TABLE 16 Percent Inhibition of Human IL-23 Mediated Increases in SerumConcentrations of Mouse IL-17F or AF by i.p. bAbs, Relative to theConcentrations of Vehicle-Treated Mice (n = 3 per Group) % Inhibition ofIL- % Inhibition of IL- 23-Mediated Serum 23-Mediated Serum mIL-17FLevels mIL-17AF Levels Vehicle (PBS) 0 0  1 mg/kg bAb1 49 22  5 mg/kgbAb1 99 94 12 mg/kg bAb1 96 94  1 mg/kg bAb2 21 17  5 mg/kg bAb2 82 7112 mg/kg bAb2 67 97  1 mg/kg bAb3 0 45  5 mg/kg bAb3 38 91 12 mg/kg bAb365 95  1 mg/kg bAb4 0 62  5 mg/kg bAb4 27 74 12 mg/kg bAb4 49 97

EXAMPLE 6 VCVFc Bispecific Antibodies

Construction and Expression of Mammalian VCVFc Bispecific Molecules

Whole genes were synthesized at GenScript (GenScript, Piscataway, N.J.,USA) and inserted into pTT5, an HEK293-6E transient expression vector(NCR Biotechnology Research Institute, Ottawa, ON, CAN) via restrictionenzyme cloning. Most constructs were expressed using the mod2610 (SEQ IDNO:30) signal sequence.

The VCVFc is a bispecific antibody which contains a whole antibody witha Fv unit of the second arm of the bispecific inserted between the Fabregion and the hinge via a linker (for example, but not limited to, 10mer G₄S for either chain, or RTVAAPS (SEQ ID NO:85) for the light chainand SSASTKGPS (SEQ ID NO:86) for the heavy chain). An illustration of aVCVFc bispecific antibody is shown in FIG. 5.

The HEK293-6E suspension cells were transfected with expressionconstructs using polyethylenimine reagent and cultivated in F17 medium(Invitrogen, Grand Island, N.Y., USA) with the addition of 5 mML-glutamine and 25 μg/mL G418. After 24 hours, 1/40th volume of 20%Tryptone NI (Organotechnie SAS, La Courneuve, FR) was added. Atapproximately 120 hours post transfection, conditioned media washarvested and passed through a 0.2 μm filter. Protein was purified fromthe filtered conditioned media using a combination of Mab Select SuReAffinity Chromatography (GE Healthcare, Piscataway, N.J., USA) andSUPERDEX® 200 Size Exclusion Chromatography (GE Healthcare, Piscataway,N.J., USA). Content was estimated by absorbance at UV-A280 nm andquality evaluated by analytical size exclusion high performance liquidchromatography, SDS PAGE, and western blot.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity;NIH/3T3/KZ170 NF-κB Luciferase Reporter Assay to Measure Human IL-17A,IL-17A/F, and IL-17F Activity by NF-κB Induction

The bioassay was performed as described in Example 1 hereinabove.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity;Baf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay to MeasureHuman IL-23Activity by Phospho-STAT3 Induction

The bioassay was performed as described in Example 3 hereinabove.

PDGF-C/PDGF-D VCVFc Bispecific Antibodies Bioassay Activity; NormalHuman Lung Fibroblasts (NHLF) Proliferation Assay to Measure HumanPDGF-C and PDGF-D Mitogenic Activity

A primary normal human lung fibroblast cell line (NHLF, CC-2512, Lonza,Walkersville, Md.) was seeded at 1,000 cells/well in growth media (FGM-2BulletKit, Lonza, Walkersville, Md.) and incubated overnight at 37° C.,5% CO₂. The following day media was removed and serial dilutions ofrecombinant human PDGF-C and PDGF-D (ZymoGenetics) were made up in assaymedia (FBM plus 0.1% BSA, Lonza, Walkersville, Md.) and added to theplates containing the cells and incubated together at 37° C., 5% CO₂ for48 hours. Additionally the assay was used to measure neutralizationPDGF-C and PDGF-D activity. A sub maximal concentration of PDGF-C orPDGF-D was combined with serial dilutions of anti-human PDGF-C/D oranti-human PDGFRα/β VCVFc antibodies described herein in assay media andadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 48 hours. Cells were pulsed with 1 μCi/well of Thymidine[Methyl-³H](PerkinElmer, Waltham, Mass.) and incubated at 37° C., 5% CO₂for an additional 24 hours. Following incubation mitogenic activity wasassessed by measuring the amount of ³H-Thymidine incorporation. Mediawas removed and cells trypsinized for 10 minutes at 37° C. before beingharvested on FilterMate harvester (Packard Instrument Co., Meriden,Conn.) and read on TOPCOUNT® microplate scintillation counter (PackardInstrument Co., Meriden, Conn.) according to manufactures instructions.Increases in ³H-Thymidine incorporation were indicative of a PDGF-C orPDGF-D receptor-ligand interaction. Decreases in ³H-Thymidineincorporation were indicative of neutralization of the PDGF-C or PDGF-Dreceptor-ligand interaction. IC₅₀ (inhibitory concentration at 50percent) values were calculated using GraphPad Prism 4 software(GraphPad Software, Inc., San Diego Calif.) for each PDGF-C/PDGF-D orPDGFRα/PDGFRβ VCVFc bispecific antibody.

IL-23/IL-17A/F VCVFc Bispecific Antibody Bioassay Activity;NIH/3T3/KZ170 NF-κB Luciferase Reporter Assay andBaf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay Results

Human IL-17A, IL-17A/F and IL-17F induce activation of the NF-κBluciferase reporter in a dose dependent manner with an EC₅₀concentration determined to be 0.15 nM for IL-17A, 0.5 nM for IL-17A/Fand 0.5 nM for IL-17F and IL-23 induces STAT3 phosphorylation in a dosedependent manner with an EC₅₀ concentration determined to be 0.02 nM.The IC₅₀ data for the anti-human IL-23/IL-17A/F VCVFc bispecificantibodies are shown below in Tables 17, 18 and 19.

IL-23/17A/F VCVFc Bispecific Antibody Table

TABLE 17 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339-134 mAbMVC978 MVC717 3 0.9 0.4 Not Done IgG1.1 SEQ ID SEQ ID NO: 64 NO: 66IL23.6 (7B7) MVC1003 MVC1002 Not Done Not Done Not Done 0.2 mAb IgG1.1SEQ ID SEQ ID NO: 68 NO: 17 23/17VCV1 MVC1020 MVC1021 20 3 3 0.008IgG1.1 SEQ ID SEQ ID NO: 87 NO: 89 23/17VCV2 MVC1022 MVC1023 0.4 0.4 0.90.3 IgG1.1 SEQ ID SEQ ID NO: 91 NO: 93

TABLE 18 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339-134 mAbMVC978 MVC717 1.2 0.23 0.28 Not Done IgG1.1 SEQ ID SEQ ID NO: 64 NO: 66IL23.6 (7B7) MVC1003 MVC1002 Not Done Not Done Not Done 0.0030 mAbIgG1.1 SEQ ID SEQ ID NO: 68 NO: 17 23/17VCV3 MVC1119 MVC1021 16 9.7 6.00.011 IgG4.1 SEQ ID SEQ ID NO: 95 NO: 89 23/17VCV4 MVC1120 MVC1023 0.200.34 0.20 0.47 IgG4.1 SEQ ID SEQ ID NO: 97 NO: 93 23/17VCV5 MVC1122MVC1121 15 8.7 7.0 0.0038 IgG1.1 SEQ ID SEQ ID NO: 99 NO: 101 23/17VCV6MVC1124 MVC1123 0.38 0.35 0.29 0.043 IgG1.1 SEQ ID SEQ ID NO: 103 NO:105

TABLE 19 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339.15.3.6N/A N/A 9.8 0.34 0.32 Not Done mAb Hybridoma line lot E10915 IL23.4 mAbSEQ ID SEQ ID Not Done Not Done Not Done 0.029 IgG4.1 - BDC NO: 107 NO:109 Lot PC-1413- 32 23/17VCV7 MVC1108 MVC1107 24 13 5.9 0.053 IgG1.1 SEQID SEQ ID NO: 111 NO: 113 23/17VCV8 MVC1110 MVC1109 2.4 0.34 0.31 2.6IgG1.1 SEQ ID SEQ ID NO: 115 NO: 117PDGF-C/PDGF-D and PDGFRα/PDGFβ VCVFc Bispecific Antibodies BioassayActivity; Normal Human Lung Fibroblasts (NHLF) Proliferation AssayResults

PDGF-C and PDGF-D induce proliferation of the NHLF cells in a dosedependent manner with a sub maximal concentration determined to be 0.1nM for PDGF-C and 6 nM for PDGF-D. Table 20 and Table 21 present IC₅₀data for the PDGF-C/PDGF-D or PDGFRα/PDGFRβ VCVFc bispecific antibodydescribed herein.

PDGF-C/PDGF-D VCVFc Bispecific Antibody Table

TABLE 20 Heavy Chain Light Chain MVC# MVC# PDGFC PDGFD Name SEQ ID NO:SEQ ID NO: IC₅₀ nM IC₅₀ nM PDGFC mAb N/A N/A  .083 Not Hybridoma DoneLot-E2826 PDGFD mAb N/A N/A Not Done 3.5 Hybridoma Lot-E4342 C/DVCV1MVC1112 MVC1111 0.090 20 IgG1.1 SEQ ID NO: 119 SEQ ID NO: 121PDGFRα/PDGFRβ VCVFc Bispecific Antibody Table

TABLE 21 Heavy Chain Light Chain MVC# MVC# PDGFC PDGFD Name SEQ ID NO:SEQ ID NO: % Inhibition % Inhibition PDGFRα mAb N/A N/A 100% 30%Hybridoma Lot-C5161 PDGFRβ mAb N/A N/A 50% 100% Hybridoma Lot-C8938α/βVCV2 MVC1118 MVC1117 70% 100% IgG1.1 SEQ ID SEQ ID NO: 123 NO: 125IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity; PrimaryHuman Fibroblast Assay to Measure Human IL-17A, IL-17A/F, and IL-17FActivity by IL-6 Induction

The bioassay was pertextured as described in Example 3 hereinabove.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity; PrimaryHuman Fibroblast Assay Results

Human IL-17A, IL-17A/F and IL-17F induce human IL-6 production in a dosedependent manner with an EC₅₀ concentration determined to be 0.08 nM forIL-17A, 25 nM for IL-17A/F and 25 nM for IL-17F. Anti-humanIL-23/IL-17A/F VCVFc bispecific antibody 23/17VCV2 (SEQ ID NO:91 and SEQID NO:93). Table 22 presents example IC₅₀ data for the IL-23/IL-17A/FVCVFc bispecific antibody described herein.

TABLE 22 23/17VCV2 339-134 IL23.6 (7B7) IgG1.1 mAbIgG1.1 mAbIgG1.1 SEQID NO: 91 SEQ ID NO: 64 SEQ ID NO: 68 Profile SEQ ID NO: 93 SEQ ID NO:66 SEQ ID NO: 17 Cellular Potency IL-17A 0.3 nM  2 nM Not Done Hu.primary EC₅₀ = 0.08 nM fibroblast cells IL-17AF  26 nM 22 nM Not Done(HFFF) EC₅₀ = 25 nM IC₅₀ IL-17F  25 nM 23 nM Not Done EC₅₀ = 25 nMCellular potency IL-23 0.4 nM Not Done 0.02 nM Primary T cell EC₅₀ =0.02 nM assay IC₅₀IL-23/IL-17A/F VCVFc Bispecific Antibodies Co-Binding Activity; PrimaryHuman Fibroblast Assay to Measure the Inhibition of Human IL-17A,IL-7A/F, or IL-F while Simultaneously Bound to Human IL-23. The PrimaryHuman T Cell Phospho-STAT3 Assay to Measure the Inhibition of HumanIL-23 while Simultaneously Bound to Human IL-17A, IL-7A/F, or IL-17F.

The primary human fibroblast assay was run in the presence of excessamounts of IL-23 at 30 nM. The primary human T cell phospho-STAT3 assaywas run in the presence of excess amounts of IL-17A, IL-17A/F, andIL-17F at 30 nM.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Co-Binding Results

Bispecific antibody 23/17VCV2 (SEQ ID NO:91 and SEQ ID NO:93) whenexamined in the presence of human IL-23 did not interfere with humanIL-17A, IL-17A/F, IL-17F inhibition. Bispecific antibody 23/17VCV2 whenexamined in the presence of human IL-17A, IL-17A/F, IL-17F did notinterfere with human IL-23 inhibition.

Measurement of Binding Affinities of IL-23/IL-17A/F VCVFc BispecificAntibodies to Human IL-17A, IL-17A/F, IL-17F, and Human IL-23 ViaSurface Plasmon Resonance (Biacore)

Binding activities were determined as described in Example 3hereinabove.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Biacore Activity

The results of the binding experiments with human IL-17A, IL-17A/F, andIL-17F are shown in Tables 23, 24, and 25, respectively. The results ofthe binding experiments with the human IL-23/IL-12B heterodimer areshown in Table 26.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity for IL-17A

TABLE 23 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)K_(D1) (nM) 339-134 mAb  2.E+06  3.E−03  1.E−9 1.0 IgG1.1 SEQ ID NO: 64SEQ ID NO: 66 23/17VCV2 3.8E+06 3.4E−03 8.9E−10 0.9 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 5.4E+06 5.4E+03 1.0E+09 1.0 IgG4.1 SEQ ID NO: 97SEQ ID NO: 93 23/17VCV6 4.0E+06 4.7E+03 1.2E+09 1.2 IgG1.1 SEQ ID NO:103 SEQ ID NO: 105IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity for IL-17A/F

TABLE 24 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)K_(D1) (nM) 339-134 mAb  2.E+06  5.E−04  2.E−10 0.2 IgG1.1 SEQ ID NO: 64SEQ ID NO: 66 23/17VCV2 1.8E+06 7.1E−04 3.9E−10 0.4 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 1.5E+06 7.7E+04 5.1E+10 0.5 IgG4.1 SEQ ID NO: 97SEQ ID NO: 93 23/17VCV6 1.2E+06 7.9E+04 6.6E+10 0.7 IgG1.1 SEQ ID NO:103 SEQ ID NO: 105IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity for IL-17F

TABLE 25 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)K_(D1) (nM) 339-134 mAb  2.E+06  2.E−04  1.E−10 0.1 IgG1.1 SEQ ID NO: 64SEQ ID NO: 66 23/17VCV2 2.4E+06 5.1E−04 2.1E−10 0.2 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 2.2E+06 3.5E+04 1.6E+10 0.2 IgG4.1 SEQ ID NO: 97SEQ ID NO: 93 23/17VCV6 3.4E+06 1.2E+04 3.5E+11 0.04 IgG1.1 SEQ ID NO:103 SEQ ID NO: 105IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity forIL23/IL-12B

TABLE 26 Antibody or k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹)K_(D1) (M) K_(D1) (nM) 7B7Mab  3.E+05  2.E−04  7.E−10 0.7 SEQ ID NO: 68SEQ ID NO: 17 23/17VCV2 4.7E+04 2.1E−04 4.5E−09 4.5 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 No No Binding No No Binding IgG4.1 BindingBinding SEQ ID NO: 97 SEQ ID NO: 93 23/17VCV6 5.6E+04 1.1E+04 2.0E+092.0 IgG1.1 SEQ ID NO: 103 SEQ ID NO: 105Simultaneous Co-Binding of IL-17A/F and IL-23 to the IL-23/IL-17A/FVCVFc Bispecific Antibodies Via Surface Plasmon Resonance (Biacore)

This assay was performed as described in Example 3 hereinabove.

Simultaneous Co-Binding of IL-17A/F and IL-23 to the IL-23/IL-17A/FVCVFc Bispecific Antibodies Via Surface Plasmon Resonance (Biacore)Results

Bispecific antibody 23/17VCV2 (SEQ ID NO:91 and SEQ ID NO:93) was ableto simultaneously co-bind both human IL-23 and human IL-17A/F,demonstrating that both arms of the bispecific antibodies werefunctional.

EXAMPLE 7 IL-23p19 Epitope Mapping

The analysis described in this Example 7 aims to identify the epitopicresidues on IL-23p19 for which the IL-23p19 antibody (7B7 antibody orMab, 7B7 Fab and biAb3, all of which have a heavy chain variable domainas shown in SEQ ID NO:7 and light chain variable domain as shown in SEQID NO:9) binds. Fab 7B7, 7B7 antibody and biAb3 have all been used inthe binding studies at various stages because they are interchangeableas far as their epitope on IL-23p19.

Proteolytic Digest and Peptide Data on Epitope

Mass spectrometry epitope sequence analysis of the IL-23p19 antibody wasbased on both epitope extraction and epitope excision methods. (Parkeret al., “MALDI/MS-based epitope mapping of antigens bound to immobilizedantibodies”, Mol. Biotechnol., 20(1):49-62 (January 2002)). In bothcases the IL-23p19 antibody was directly immobilized via primary aminesof the antibody on surface-activated beads at an average density of 2 mgmAb per 1 ml bed volume. Peptides from IL-23 his-tag antigen weregenerated with or without reduction and alkylation. Reduction of theantigen IL-23 was performed by incubating with 50 mM dithiothreitol inPBS and 4M guanidine HCl for 1 hour at 37° C. This was followed byalkylation with 100 mM iodoacetamide for 30 minutes at room temperature.Reduced and alkylated IL-23 was dialyzed against PBS overnight prior tofragmentation. For epitope extraction, antigen peptides were generatedby proteolytic digestion with the endoproteinases trypsin, chymotrypsin,lys-C, arg-C, asp-N and or glu-C with an enzyme to antigen ratio of upto 2% (w/w). Incubations were performed at 37° C. with incubation timesranging from 2 hours to overnight. The resulting peptides were mixedwith antibody resin at room temperature for 30 minutes. This resin wasthen washed three times to remove any non-specifically bound peptides.All digestion, incubation, and wash steps were performed in PBS pH 7.The same protocol was followed for epitope excision except that theintact antigen was incubated with the antibody for 30 minutes at roomtemperature prior to enzymatic digestion. In both methods antibody boundpeptides were eluted and analyzed on ESI-MS.

These data indicate that IL-23p19 antibody has a discontinuous epitopecomprised of three peptide regions in IL-23p19. Synthetic peptides weregenerated to further examine these three peptide regions, and theirbinding was tested and analyzed by both ELISA and mass spectrometry.Based on these observations, it is suggested that the following peptidesrepresent the sequences of the IL-23p19 antibody epitope:

Peptide 1: (residues 156-167 of SEQ ID NO: 6) WQRLLLRFKILR Peptide 2:(residues 46-57 of SEQ ID NO: 6) SAHPLVGHMDLR Peptide 3:(residues 93-117 of SEQ ID NO: 6) IHQGLIFYEKLLGSDIFTGEPSLLP. IL-23 Epitope Mapping by HDX-MS

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) method probesprotein conformation and conformational dynamics in solution bymonitoring the rate and extent of deuterium exchange of backbone amidehydrogen atoms. The level of HDX depends on the solvent accessibility ofbackbone amide hydrogen atoms and the conformation of the protein. Themass increase of the protein upon HDX can be precisely measured by MS.When this technique is paired with enzymatic digestion, structuralfeatures at the peptide level can be resolved, enabling differentiationof surface exposed peptides from those folded inside. Typically, thedeuterium labeling and subsequent quenching experiments are performed,followed by online pepsin digestion, peptide separation, and MSanalysis. Prior to epitope mapping of BMS-986113 in IL-23 by HDX-MS,non-deuteriated experiments were performed to generate a list of commonpeptic peptides for IL-23 (4.4 mg/mL) and IL-23/BMS-986113 (1:1 molarratio, 4.4 mg/mL & 3.36 mg/mL), achieving a sequence coverage of 97% forIL-23. In this experiment, 10 mM phosphate buffer (pH 7.0) was usedduring the labeling step, followed by adding quenching buffer (200 mMphosphate buffer with 1.5M GdnC1 and 0.5M TCEP, pH 2.5, 1:1, v/v). Forepitope mapping experiments, 5 μL of each sample (IL-23 orIL-23/BMS-986113 (1:1 molar ratio)) was mixed with 65 μL HDX labelingbuffer (10 mM phosphate buffer in D₂O, pD 7.0) to start the labelingreactions at room temperature (˜25° C.). The reactions were carried outfor different periods of time: 20 sec, 1 min, 10 min, 60 min and 240min. By the end of each labeling reaction period, the reaction wasquenched by adding quenching buffer (1:1, v/v) and the quenched samplewas injected into Waters HDX-MS system for analysis. The observed commonpeptic peptides were monitored for their deuterium uptake levels in theabsence/presence of BMS-986113. The same protocol was followed forepitope mapping of anti-IL-23 7B7 Fab (4.91 mg/mL) in IL-23 by HDX-MS.

Epitope mapping of anti-IL-23 7B7 Fab in complex with IL-23 and biAb3with IL-23 indicate that biAb3 has a discontinuous epitope comprised offive peptide regions in IL-23p19. Based on relative deuterium uptakelevels, five peptide regions can be ranked as region 1>2>3>4>5 withregion 1 having the most significant changes in deuterium uptakes andregion 5 having the least significant changes in deuterium uptakes. Thefive peptide regions on IL-23p19 as determined by HDX-MS for theIL-23p19 antibody were determined as follows:

Region 1: (residues 117-124 of SEQ ID NO: 6) PDSPVGQL; Region 2:(residues 108-116 of SEQ ID NO: 6) IFTGEPSLL; Region 3:(residues 164-172 of SEQ ID NO: 6) KILRSLQAF; Region 4:(residues 34-55 of SEQ ID NO: 6) QQLSQKLCTLAWSAHPLVGHMD; and Region 5:(residues 89-105 of SEQ ID NO: 6) CLQRIHQGLIFYEKLLG.Computational Epitope Prediction and Design of Alanine Shave Mutants

Alanine shave mutagenesis is a strategy for mutating multiple residuesin the same construct to alanine to remove the amino acid side chains inepitope of binding (Wells, J. A., “Systemic mutational analyses ofprotein-protein interfaces”, Enzym., 202:390-411 (1991)). Multiplesources of information on the involvement of residues in a potentialepitope with the 7B7 Fab and biAb3 were combined to produce a targetedlist of regional alanine shave mutants. The residues contained in theoverlapping regions between both the HDX (see above in this Example 7)and the proteolytic digest peptide mapping (see above in this Example 7)were mapped onto the sequence of the IL-23p19 domain and three linearregions of common residues were identified as Regions A, B and C. RegionA corresponds to amino acid residues 33-59 of SEQ ID NO:6. Region Bcorresponds to amino acid residues 89-125 of SEQ ID NO:6. Region Ccorresponds to amino acid residues 144-173 of SEQ ID NO:6. In order tocalculate the residues whose side chains are exposed (solvent accessiblesurface area, SASA) and would therefore be located on the proteinsurface of the p19 domain of IL-23, an in-house structure of the IL-23heterodimer was used. For each residue in the p19 domain of IL-23 theratio of accessible surface to the standard exposed surface for theamino acid type was calculated and residues were grouped into bins.Residues were placed in accessibility bins as follows: <30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, >90% exposed. The standard residueaccessibilities for each amino acid type were calculated in the extendedtripeptide Gly-X-Gly. The second calculation performed was ODA (OptimalDocking Area) which is useful for predicting likely protein-proteininteraction surfaces. The method identifies optimal surface patches withthe lowest docking desolvation energy. The ODA was calculated for thep19 domain of IL-23 and these results used to prioritize residues formutagenesis.

Residues in these three regions (A, B, C) were prioritized based upon ahigh score in both the ODA and SASA calculations and also weighted basedon the extent of hydrogen-deuterium exchange relative to the uncomplexedIL-23 (peptide #1>#2>#3>#4>#5). Regions with non-identity to mouse werecloned as mouse swap mutants, instead of alanine shave mutants, whichdid not show an impact on binding in small scale testing. With theexception of M7 which contains a linear sequence of residues in anextended loop, residues were then combined into non-linear epitopesbased on mapping them to the X-ray crystallographic structure of IL-23(M5, M6, M8). Additional backup mutants were generated with thesub-epitopes of predominantly linear residues (M9, M10, M11). Thealanine shave mutants designed by this method are shown below in Table27.

TABLE 27 Alanine Shave Mutants of IL-23p19 IL-23p19 Name Region Mutated(SEQ ID NO: 6) Residues Mutated to Alanine M5 A and B H53A, M54A, E112A,L116A and D118A M6 A and C T42A, W45A, H48A, F163A and Q170A M7 C W142A,E143A, T144A, Q145A and Q146A M8 A, B and C H53A, E112A, Q154A and W156AM9 B L116A, D118A and Q123A M10 A H53A, M54A, D55A and F163A M11 CW142A, T144A and Q146ACloning, Expression and Purification of IL-23 Epitope Mapping AlanineMutants

Non-tagged wild-type IL-23 p40 subunit entry vector construct wasgenerated by PCR and the fidelity of the PCR fragment was confirmed bysequencing. The transient expression construct was generated by GatewayLR recombination and sequence confirmed. The His-tagged wild-type IL-23p19 subunit construct and all mutant constructs were generated by PCRand cloned into the transient expression vector directly. The fidelityof all PCR fragments was confirmed by sequencing. To generate thewild-type control, non-tagged wild-type IL-23 P40 subunit wasco-expressed with the His-tagged wild-type P19 subunit transiently inHEK293-6E cells at 4 L scale for IL-23 complex purification. Briefly,HEK293-6E cells at 1×10⁶ cells/ml were transfected with expressionplasmids/PEI complex at the ratio of 0.5 (p19)/0.5 (p40)/1.5 (PEI).Tryptone N1 feed was added 24 hours later and cells harvested on 120hours post transfection. The conditioned media was filtered with 0.2 μMfilters. Seven His-tagged IL-23 p19 mutant constructs wereco-transfected with the non-tagged wild-type IL-23 p40 subunit at the 30ml scale following the same transfection protocol described above. Theconditioned media were transferred for analysis and the expression ofall mutants was confirmed by anti-His Western-blot. Based on preliminarybinding results, mutants M5, M7, M9, and M10 were selected and scaled upat 2 L scale with the same transfection protocol. The wild-type was alsoscaled up at 2L. The scale-ups of wild-type and mutants of IL-23 at 2liters of HEK cells were harvested and the supernatants wereconcentrated and buffer exchanged to PBS by tangential flow filtrationwith a 10 kDa membrane. The proteins were then purified by immobilizednickel affinity chromatography. The wild-type was eluted with 40 mMimidazole and then buffer exchanged by desalting gel filtrationchromatography to PBS (5.6 mM Na₂HPO₄, 1.1 mM KH₂PO₄, 154 mM NaCl, pH7.4). The purity of the wild-type was determined by SDS-PAGE to be >95%.The mutants were washed with 40 mM imidazole followed by elution at 500mM Imidazole. The elution pools were then buffer exchanged by dialysisto PBS (7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 130 mM NaCl, pH 7.1). The purity ofthe mutants was determined by SDS-PAGE to be >95%. Single alaninemutants of his-tagged IL-23p19 at key residues identified by alanineshave mutagenesis were generated by gene synthesis and then cloned intothe transient transfection vector. Expression and purification weresimilar to the alanine shave mutants with the exception of M35A whichwas an affinity purified.

Biacore Binding Analysis of IL-23 Mutants to the IL-23p19 Antibody

The binding of the 30 mL small scale expression of all seven (7) alaninemutants (see Table 27) was measured by surface Plasmon resonance (SPR,Biacore)) on a BIACORE® T100 in PBST (7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 130 mMNaCl, 0.05% Tween 20, pH 7.4) at 25° C. The relevant antibodies andreceptors were captured at a level of about 60 RUs by protein Aimmobilized at 2000 RUs on a CM5 sensor chip. In addition to the biAb3,Merck's IL-23 p19 mAb (7G10) and STELARA® (IL-12/IL-23 p40 mAb) wereused as controls for domain binding. In addition, the commercialreceptors for IL-23 were used as controls hIL-23R-Fc and hIL-12Rβ1-Fc(both from R&D Systems). The supernatants were diluted 1:5 into PBST andinjected at 30 μL/min over the mAb or receptor surface for 3 minutesand, after a dissociation time, regenerated with 10 mM Glycine, pH 2.0.Binding to a reference surface of Protein A without any capturedantibody was subtracted from all specific binding curves beforeanalysis. The results shown in FIG. 9 show the response 10 secondsbefore the end of the injection.

The Biacore results demonstrate that the IL-23 alanine shave mutantsmaintain binding to the p40 specific antibody and the p19 specific IL-23receptor (except for M8 which potentially describes the receptor bindingsite). Binding of another IL-23p19 mAb and IL-12Rβ1-Fc was alsoperformed and is consistent with the results of FIG. 9. The M5 mutantshows a dramatic loss of binding to 7B7 Mab while M9 & M10 show partialloss of binding to 7B7 Mab (see FIG. 10). M7 does not show any impact tobinding to the antibody or controls. Therefore, these four mutants werechosen for scale-up and purification, the three mutants that losebinding to the IL-23p19 antibody (7B7 antibody or Mab, 7B7 Fab andbiAb3, all of which have a heavy chain variable domain as shown in SEQID NO:7 and light chain variable domain as shown in SEQ ID NO:9) and theM7 as a control.

Biacore analysis of the purified mutants used the same assay format asthe supernatants except that the wild-type and mutant IL-23 was dilutedto 25 nM and serially titrated 1:2 down to 1.5 nM. All results obtainedwith the purified IL-23 mutants confirmed the data obtained with theexpression supernatants. The data were fit to a 1:1 Langmuir bindingmodel to determine the Kd values shown in FIG. 10 and Table 28. The 1-2RU of reference subtracted binding observed for M5 binding to theIL-23p19 antibody (7B7 antibody or Mab, 7B7 Fab and biAb3, all of whichhave a heavy chain variable domain as shown in SEQ ID NO:7 and lightchain variable domain as shown in SEQ ID NO:9) was simulated using theBIAsimulation software 2.1 using the average Rmax determined fromkinetic analysis of M9 & M10. The affinity was estimated to be ≧330 μMwith a 1500 fold weaker Kd than wild-type as shown in Table 28.

TABLE 28 Biacore Kinetic Analysis of IL-23 Alanine Mutants BindingIL-23p19 Antibody Merck 7B7 mab BiAb3 STELARA ® 7G10 Kd Kd-shift ΔΔG ΔΔGΔΔG ΔΔG Variant (nM) (from WT) (kcal/mole) (kcal/mole) (kcal/mole)(kcal/mole) WT 0.14 NA NA NA NA NA M5 ≧300 1500 4.6 3.8 0.1 1.9 M7 0.4 30.5 NM 0.1 0.2 M9 25 140 2.9 2.3 0.1 0.2 M10 43 230 3.2 2.3 0.3 0.4

Biacore analysis of single alanine mutants was performed to confirm thenon-linear epitope demonstrated by the M5, M9, and M10 alanine shavemutants. Most single alanine mutants in the three linear regions A, B,and C showed no change in binding to the 7B7 mAb or biAb3. Only three ofthe fourteen single alanine mutant of IL-23 tested showed a significantdecrease in binding affinity greater than 1 kcal/mole. The affinity andthe AAG values for the key residues overlapping between alanine shavemutants M5, M9, and M10 shown in Table 29 and demonstrate that one majorresidue in linear region B and two residues in linear region Acontribute predominantly to the binding energy of the IL-23p19antibody-IL-23 complex.

TABLE 29 Biacore Kinetic Analysis of IL-23 Single Alanine MutantsBinding IL-23p19 Antibody Merck 7B7 mab BiAb3 STELARA ® 7G10 Kd Kd-shiftΔΔG ΔΔG ΔΔG ΔΔG Variant (nM) (from WT) (kcal/mole) (kcal/mole)(kcal/mole) (kcal/mole) WT 0.14 NA NA NA NA NA His53 0.2 0 0.2 0.2 0.3 0(region A) Met 54 6.8 48 2.3 2.2 0.3 1.0 (region A) Asp55 9.6 68 2.5 2.30.5 0.7 (region A) Glu112 0.7 5 0.9 1.2 0.3 1.7 (region B) Leu116 46 3303.4 3.0 0.5 0.2 (region B) Asp118 0.1 0 0 −0.4 −0.3 −0.3 (region B)IL-23 Induced STAT3 Phosphorylation in BaF3/huIL-23Rα/huIL-12Rβ1Transfectants

A murine bone marrow derived cell line (BaF3) was stably transfectedwith human IL-23Rα and human IL-12Rβ1 full length receptors and cloned.IL-23 induction of phosphorylation of STAT3 was monitored by ELISA forIL-23 Alanine shave mutants. BaF3/huIL-23Rα/huIL-12Rβ1 (clone 6) cellswere washed three times with assay media before being plated at 50,000cells per well in 96-well round-bottom tissue culture plates.BaF3/huIL-23Rα/huIL-12Rβ1 cells respond to IL-23 in a dose dependantmanner by phosphorylation of STAT3. To assess antibody inhibition ofIL-23 signaling, an EC₅₀ concentration of 20 pM IL-23 was premixed withthree-fold serial dilutions of each antibody from 33 nM to 0.56 pM andincubated at 37° C. for 15 minutes in assay media prior to addition tothe cells. Following pre-incubation, treatments were added in duplicateto plates containing cells and incubated at 37° C. for 15 minutes tostimulate phosphorylation of STAT3. Stimulation was stopped with theaddition of ice-cold wash buffer and cells lysed according tomanufacturer's instructions (Bio-Rad Laboratories Cell Lysis kit, Cat#171-304012). Phosphorylated STAT3 levels were determined by ELISA(Bio-Rad Laboratories Phospho-STAT3^((Tyr705)) kit, Cat #171-V22552)according to manufacturer's instructions. Data was analyzed and IC₅₀values were calculated using GraphPad Prism 4 software. All the IL-23Alanine shave mutants and all the IL-23 Alanine single mutants areactive and equal potent as wt IL-23 (untagged and tagged) at inducingpSTAT3 activity on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants (Table 30).Results for biAb3, STELARA® (IL-12/IL-23 p40 mAb), and Merck's IL-23p19antibody (7G10) inhibition of IL-23 Alanine shave mutant induced pSTAT3are shown in Table 31. biAb3 neutralizes the biological activity of wtIL-23 and IL-23 M7 Alanine shave mutant with equal potency, M9 and M10with reduced potency, and does not neutralize the biological activity ofM5 on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants. STELARA® IL-12 p40 mAbneutralizes the biological activity of wt IL-23 and all the IL-23Alanine shave mutants with equal potency on BaF3/huIL-23Rα/huIL-12Rβ1transfectants. Positive control Merck IL-23 p19 mAb (7G10) neutralizesthe biological activity of wt IL-23, M7, M9 and M10 Alanine shavemutants with equal potency and does not neutralize the biologicalactivity of M5 on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants.

TABLE 30 EC₅₀ values for IL-23 Alanine shave mutants and IC₅₀ Values forbiAb3, STELARA ® (IL-12/IL-23 p40 mAb), and Merck's IL-23p19 antibody(7G10) inhibition of IL-23 Alanine shave mutants induced pSTAT3 inBaF3/huIL-23Rα/huIL-12Rβ1 transfectants Antibody wt human IL-23 IL-23 M5IL-23 M7 IL-23 M9 IL-23 M10 None (EC₅₀) 21 pM 26 pM 21 pM 33 pM 19 pMbiAb3 19 pM NA 17 pM 2400 pM  5300 pM  STELARA ® 79 pM 62 pM 59 pM 67 pM71 pM Merck 7G10 380 pM  NA 310 pM  260 pM  350 pM 

Single IL-23p19 mutations (H53A, M54A and D55A) were constructed andIC₅₀ Values for 7B7, STELARA® (IL-12/IL-23p40 mAb), and Merck's IL-23p19antibody (7G10) (Table 31) to inhibit the single IL-23p19 mutatedpolypeptides to induce pSTAT3 in BaF3/huIL-23Rα/huIL-12Rβ1 transfectantswas determined. The 7B7 mAb neutralizes the biological activity of IL-23Alanine single mutants H53A, E112A, and D118A with equal potency, M54Aand D55A mutants with significantly reduced potency, and does notneutralize the biological activity of L116A mutant compared to wt IL-23on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants (Table 31). STELARA® IL-12p40 mAb neutralizes the biological activity of all the IL-23 Alaninesingle mutants with equal potency compared to wt IL-23 onBaF3/huIL-23Rα/huIL-12Rβ1 transfectants. Merck IL-23 p19 mAb neutralizesthe biological activity of all the IL-23 Alanine single mutants withequal potency compared to wt IL-23 on BaF3/huIL-23Rα/huIL-12Rβ1transfectants, except for IL-23 E112A mutant which it does notneutralize.

TABLE 31 IC₅₀ Values for 7B7, STELARA ® (IL-12/IL-23p40 mAb), andMerck's IL-23p19 Antibody (7G10) to Inhibit IL-23p19 Single Mutations(H53A, M54A and D55A) to Induce pSTAT3 in BaF3/huIL-23Rα/huIL-12Rβ1Transfectants IL-23 IL-23 IL-23 IL-23 IL-23 IL-23 IL-23 wt H53A M54AD55A E112A L116A D118A IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ Antibody (nM)(nM) (nM) (nM) (nM) (nM) (nM) 7B7/biAb3 0.020 0.015 0.71 1.2 0.025 >30.012 STELARA ® 0.067 0.078 0.081 0.077 0.033 0.054 0.052 p40 Merck 7G100.18 0.19 0.27 0.18 >3 0.24 0.22

Taking all these results together, 7B7 mAb inhibition of IL-23 requiresamino acids residues M54, D55, and L116, but does not require amino acidresidues H53, E112, or D118. STELARA® and Merck antibodies were ableinhibit the IL-23 Alanine single mutants M54A, D55A and L116A with noloss in activity, suggesting the loss in activity seen with IL-23 M54,D55 and L116 Alanine mutants are specific to the 7B7 mAb and the biAb3.

Oligomeric State Analysis of IL-23 Mutants and 7B7 Fab Complexes

To confirm the oligomeric state of the heterodimeric IL-23 mutants andtheir ability to complex with the 7B7 Fab, the proteins were studied byanalytical size-exclusion chromatography (SEC-MALS) separation using anAGILENT® 1100 series HPLC fitted with a diode-array absorbance detector,on a Shodex Protein KW-803 column in buffer (0.1 um filtered) containing200 mM K₂PO₄ (pH 6.8 with HCl), 150 mM NaCl, and 0.02% sodium azide, ata flow rate of 0.5 mL/min. A Wyatt Technology MINIDAWN™ TREOS® laserlight scattering instrument was plumbed downstream from the HPLC,followed by a Wyatt OPTILAB® T-REX™ differential refractometer. Sixty(60) μg of each IL-23 sample was injected after filtering at aconcentration of 10.3 μM. For complex formation, 60 μg of the 7B7 Fabwas premixed with a 6% molar excess and incubated with the IL-23 proteinat a concentration of 10.9 μM at room temperature for at 3-6 hoursbefore chromatographic separation. Particulates were removed fromprotein samples with a spin filter (NANOSEP® MF, 0.2 μm, PallCorporation) prior to injection. Data were analyzed with ASTRA® 6(Wyatt) and Chemstation (Agilent). All mutants were mostly monomeric,similar to the wild-type. The M5 mutant did not show significant complexformation after pre-incubation with the Fab and eluted close to wherethe M5 IL-23 alone elutes demonstrating that little if any complex wasformed in the 10 μM concentration range of this experiment. M7 complexedand eluted similar to the wild-type. The M9 and M10 mutants form complexat these concentrations, but the mass was somewhat less than that of thewild-type and the retention time was slightly later than the wild-typeand M7, suggesting that their affinity for 7B7 was weaker than thewild-type and M7. SEC-MALS analysis of the 14 single alanine mutantsshowed that all were mostly monomeric, similar to the wild-type, andonly the L116A mutant showed a late-shifted elution time and a slightreduction in the expected mass of the complex suggesting that theaffinity of the IL-23 for the 7B7 Fab was reduced. These results areconsistent with the Biacore shift in Kd for these L116A. The effect ofthe reduced affinity of the complex of D54A and M55A with the Fab wasnot able to be resolved under the conditions of this assay.

Differential Scanning Calorimetry of IL-23 Mutants

The thermal stability profile for the wild type and mutant IL-23heterodimers was measured by differential scanning calorimetry (DSC)using a MICROCAL® VP-capillary DSC instrument. 0.7 mg/ml protein samplesin PBS (7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 130 mM NaCl, pH 7.1) were scannedfrom 10-100° C. at 90°/hr, and the resulting thermograms were subjectedto a buffer blank subtraction and fitting procedure. The denaturation ofeach molecule was characterized by two unfolding transitions and thefitted transition midpoint (Tm) values of each transition were within1-4° C. of the wild type. The results show that none of the alanineshave mutants nor the 14 single alanine mutants show significant thermaldestabilization relative to the wild type.

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis of IL-23Mutants

Secondary structure comparison of the alanine shave mutants and wildtype of IL-23 was performed using a FT-IR spectroscopy on Biotools ProtaFT-IR instrument with CaF₂ windows with a pathlength of ˜7 μm and aNe—He laser at 632.8 nm. Data were collected with a resolution of 2 cm⁻¹and analyzed with Prota/Bomem-GRAMS/31 AI software. Duplicatemeasurements were made for each sample and the method variability isabout 3%. Secondary structure content was calculated using Amide I peakas it is structure sensitive. Approximately an equal quantity of α-Helixand β-sheet was observed in all IL-23 samples as indicated by peaks at1637 cm⁻¹ for α-Helix and peak at 1637 cm⁻¹ as well as shoulder at 1687cm⁻¹ for β-sheet. Overall no significant difference in FT-IR spectrumand calculated secondary structure result was observed in the alanineshave mutants compared to the wild type IL-23.

Circular Dichroism (CD) Analysis of IL-23 Mutants

Secondary structure comparison of the alanine shave mutants and wildtype of IL-23 using CD spectroscopy was performed using a Jasco J-815Spectrophotometer. The spectra were collected at 0.25 mg/mL IL-23protein concentration in PBS pH7.1 using a 1 mm path length cells at 25°C. from 300-190 nm with a data interval of 0.1 nm, a 50 nm/min scanningspeed, a 1 nm bandwidth, and 2 accumulations. Overall no significantdifference in secondary structure profile was observed in the alanineshave mutants compared to the wild type IL-23 using circular dichroism.

Nuclear Magnetic Resonance (NMR) Spectroscopy Analysis of IL-23 Mutants

Proton NMR is a highly sensitive technique that allows one to assess theconformational state at atomic detail. 1D ¹H NMR spectra were acquiredfor each of four mutant (M5, M7, M9, M10) and wild type IL23 proteins.All proteins were dialyzed simultaneously against NMR buffer (PBS in 8%D₂O/92% H₂O) to eliminate potential differences resulting from samplepreparation. In addition, ¹H signal intensities were corrected fordifferences in protein concentration by normalizing to the wild typespectrum. All NMR data was collected at 32° C. on a Bruker Avance 3spectrometer operating at 600 MHz. 1D NMR spectra were acquired usingthe standard bruker pulse sequence (ZG) optimized for solvent andexcipient suppression using the Watergate, WET, and Water flipbackselective excitation pulse schemes. Two thousand forty-eight (2048)scans were signal averaged for each spectrum. Cosine squared apodizationwas applied prior to Fourier transformation, and a first orderpolynomial baseline correction was used to flatten the appearance of thebaseline. Examination of each of the spectra for the individual proteinsreveals that each mutant protein was properly folded, as evidenced bythe well-dispersed resonances in both the high field (<0.5 ppm) and lowfield (>6.5 ppM) regions of the spectra. The high field methylresonances indicated the presence of an intact hydrophobic core; thedownfield amide protons reflected the existence of well-formed secondarystructure (alpha helices and beta sheets). Comparison of the spectrawith that for wild type IL23 indicated a very close match, precludingthe existence of large conformational changes in the protein structureinduced by the amino acid substitutions. In addition, the NMR resultsalso indicated that extra loss in activity in MS is unlikely due to anextra large disruption in structural integrity at the mutation sites inMS. The fact that MS was considerably closer to the wild-type protein byprinciple component analysis than M9 suggests, that the followingMS-mutations which are missing in M9, i.e., H53A, MS4A and E112A do notcause much of a disruption in the MS-structure. It was also observedthat mutants M7 & M9 which contain the elimination of an aromaticresidue appeared most similar to each other in the principle componentanalysis.

Summary of IL-23 Epitope Analysis

The methods of Alanine Shave and Single Mutagenesis have been used tomap the epitope of hIL-23 for the 7B7 Fab contained in both the 7B7 Fab,7B7 antibody and biAb3. The targeted mutagenesis strategy was performedusing epitope information generated by proteolytic digest LCMS analysis,peptide binding, hydrogen/deuterium exchange mass spectrometry, solventaccessible surface area calculations and docking algorithm calculations.Small scale screening by SPR and scale-up of select purified mutantsdemonstrated a specific epitope for 7B7/biAb3 binding hIL-23. The M5mutant shows dramatic decrease in binding to 7B7/biAb3, whilemaintaining functional activity, binding to p40 and p19 specificreagents, monomeric oligomeric state, thermal stability, and secondarystructural features similar to the wild-type. The M5 mutant showed adramatic loss of binding to 7B7/biAb3 while M9 & M10, which eachcontained two of the same mutated residues as M5, each show partial lossof binding to 7B7/biAb3. The affinity of the M5 IL-23 mutant was >300 nMfor 7B7/biAb3 which is approximately 1,500-fold weaker than wild-typeIL-23 demonstrating that the residues in the M5 mutant define theepitope for 7B7/biAb3 binding to IL-23. Thus, the data suggests that the7B7/biAb3 antibody binds to a discontinuous epitope on the p19 subunitof IL-23. This discontinuous epitope on IL-23p19 comprises at least twoepitope regions (region A (amino acid residues 33-59 of SEQ ID NO:6) andregion B (amino acid residues 89-125 of SEQ ID NO:6)). Specifically,with respect to the first epitope or region A of IL-23p19, amino acidresidues 54 (Met) and 55 (Asp) of SEQ ID NO:6 contribute a significantamount of binding energy to 7B7/biAb3's ability to bind IL-23p19. Withrespect to the second epitope or region B of IL-23p19, amino acidresidue 116 (Leu) is the primary residue within Region B energeticallycontributing to 7B7/biAb3's ability to bind IL-23p19.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. An isolated nucleic acid molecule encoding abispecific antibody comprising an IL-17A/F binding entity and an IL-23binding entity, wherein the IL-23 binding entity comprises two pairs ofimmunoglobulin chains, each pair having one light and one heavy chain,which light chain variable domain comprises a CDR1 having the amino acidsequence of SEQ ID NO:22, a CDR2 having the amino acid sequence of SEQID NO:23, and a CDR3 having the amino acid sequence of SEQ ID NO:24, andheavy chain variable domain comprises a CDR1 having the amino acidsequence of SEQ ID NO:19, a CDR2 having the amino acid sequence of SEQID NO:20, and a CDR3 having the amino acid sequence of SEQ ID NO:21, andwherein the IL-17A/F binding entity comprises two Fab fragments whichare linked to the C-terminus of the heavy chain of the IL-23 bindingentity, wherein each IL-17A/F Fab comprises a light chain variabledomain comprising a CDR1 having the amino acid sequence of SEQ ID NO:22,a CDR2 having the amino acid sequence of SEQ ID NO:23, and a CDR3 havingthe amino acid sequence of SEQ ID NO:24; and a heavy chain variabledomain comprising a CDR1 having the amino acid sequence of SEQ ID NO:25,a CDR2 having the amino acid sequence of SEQ ID NO:26, and a CDR3 havingthe amino acid sequence of SEQ ID NO:27.
 2. The isolated nucleic acidmolecule of claim 1, wherein both light chain variable domains of theIL-17A/F binding entity and the IL-23 binding entity comprise the aminoacid sequence of SEQ ID NO:9.
 3. The isolated nucleic acid molecule ofclaim 1, wherein the light chain constant domain of the IL-17A/F bindingentity and the IL-23 binding entity comprises the amino acid sequence ofSEQ ID NO:10.
 4. The isolated nucleic acid molecule of claim 1, whereinthe light chain variable domain of the IL-17A/F binding entity and theIL-23 binding entity comprises the amino acid sequence of SEQ ID NO:9and the light chain constant domain comprises the amino acid sequence ofSEQ ID NO:10.
 5. The isolated nucleic acid molecule of claim 1, whereinboth light chains of the IL-17A/F binding entity and the IL-23 bindingentity comprise the amino acid sequence of SEQ ID NO:17.
 6. The isolatednucleic acid molecule of claim 1, wherein the light chain of theIL-17A/F binding entity and the IL-23 binding entity is kappa or lambda.7. The isolated nucleic acid molecule of claim 1, wherein the heavychain variable domain of the IL-17A/F binding entity comprises the aminoacid sequence of SEQ ID NO:13.
 8. The isolated nucleic acid molecule ofclaim 1, wherein the heavy chain C_(H1) of the IL-17A/F binding entitycomprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15. 9.The isolated nucleic acid molecule of claim 1, wherein the heavy chainvariable domain of the IL-23 binding entity comprises the amino acidsequence of SEQ ID NO:7.
 10. The isolated nucleic acid molecule of claim1, wherein the heavy chain constant domain of the IL-23 binding entitycomprises the amino acid sequence of SEQ ID NO:8 or amino acid residues1-326 of SEQ ID NO:8.
 11. The isolated nucleic acid molecule of claim 1,wherein the heavy chain constant domain of the IL-23 binding entitycomprises the amino acid sequence of SEQ ID NO:11.
 12. The isolatednucleic acid molecule of claim 1, wherein the heavy chain of thebispecific antibody, which comprises the IL-23 binding entity and theIL-17A/F binding entity, comprises the amino acid sequence of SEQ IDNO:74.
 13. The isolated nucleic acid molecule of claim 1, wherein theheavy chain of the bispecific antibody comprises a human constantregion.
 14. The isolated nucleic acid molecule of claim 13, wherein theisotype of the heavy chain is IgG1, IgG2, IgG3 or IgG4.
 15. The isolatednucleic acid molecule of claim 14, wherein the IgG4 heavy chain has aSerine to Proline mutation at position 241 according to Kabat.
 16. Theisolated nucleic acid molecule of claim 1, wherein the light chain ofthe bispecific antibody comprises the amino acid sequence of SEQ IDNO:17 and the heavy chain of the bispecific antibody comprises the aminoacid sequence of SEQ ID NO:74.
 17. An expression vector comprising thefollowing operably linked elements: a transcription promoter; a nucleicacid molecule encoding the light chain of the bispecific antibody ofclaim 1, wherein the IL-23 binding entity and the IL-17A/F bindingentity comprise the same light chain amino acid sequence; and atranscription terminator.
 18. An expression vector comprising thefollowing operably linked elements: a transcription promoter; a nucleicacid molecule encoding the heavy chain of the bispecific antibody ofclaim 1, which includes the heavy chain of the IL-23 binding entity andthe IL-17A/F binding entity; and a transcription terminator.
 19. Arecombinant host cell comprising the expression vector of claim
 18. 20.The recombinant host cell of claim 19, further comprising an expressionvector comprising: a transcription promoter; a nucleic acid moleculeencoding the light chain of the bispecific antibody; wherein the IL-23binding entity and the IL-17A/F binding entity share the light chain;and a transcription terminator; and wherein the cell expresses the heavychain and light chain.
 21. A method of producing a bispecific antibodycomprising: a) culturing the host cell of claim 20, wherein theexpressed heavy and light chains form the bispecific antibody; and b)recovering the bispecific antibody.
 22. An expression vector comprisingthe following operably linked elements: a transcription promoter; afirst nucleic acid molecule encoding the heavy chain of the bispecificantibody of claim 1, which includes the heavy chain of the IL-23 bindingentity and the IL-17A/F binding entity; a second nucleic acid moleculeencoding the light chain of the bispecific antibody of claim 1, whereinthe IL-23 binding entity and the IL-17A/F binding entity comprise thesame light chain amino acid sequence; and a transcription terminator.23. A recombinant host cell comprising the expression vector of claim22.
 24. A method of producing the bispecific antibody encoded by thenucleic acid molecule in the expression vector of claim 22 comprising:a) culturing a host cell comprising said vector under conditions whereinthe encoded heavy and light chains are expressed and form the bispecificantibody; and b) recovering the bispecific antibody.
 25. An expressionvector comprising the following operably linked elements: a firsttranscription promoter; a first nucleic acid molecule encoding the heavychain of the bispecific antibody of claim 1, which includes the heavychain of the IL-23 binding entity and the IL-17A/F binding entity; afirst transcription terminator; a second transcription promoter; asecond nucleic acid molecule encoding the light chain of the bispecificantibody of claim 1, wherein the IL-23 binding entity and the IL-17A/Fbinding entity comprise the same light chain amino acid sequence; and asecond transcription terminator.
 26. A recombinant host cell comprisingthe expression vector of claim
 25. 27. A method of producing thebispecific antibody encoded by the nucleic acid molecules in theexpression vector of claim 25 comprising: a) culturing a host cellcomprising said vector under conditions wherein the encoded heavy andlight chains are expressed and form the bispecific antibody; and b)recovering the bispecific antibody.
 28. An isolated nucleic acidmolecule encoding a monoclonal antibody that specifically binds toIL-17A (SEQ ID NO:2) and IL-17F (SEQ ID NO:4) and comprises a heavychain variable domain and a light chain variable domain, wherein theheavy chain variable domain comprises a CDR1 having the amino acidsequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27, andwherein the light chain variable domain comprises a CDR1 having theamino acid sequence of SEQ ID NO:22, a CDR2 having the amino acidsequence of SEQ ID NO:23, and a CDR3 having the amino acid sequence ofSEQ ID NO:24.
 29. The isolated nucleic acid molecule of claim 28,wherein the light chain variable domain of the monoclonal antibodycomprises the amino acid sequence of SEQ ID NO:9.
 30. The isolatednucleic acid molecule of claim 28, wherein the light chain constantdomain of the monoclonal antibody comprises the amino acid sequence ofSEQ ID NO:10.
 31. The isolated nucleic acid molecule of claim 28,wherein the light chain variable domain of the monoclonal antibodycomprises the amino acid sequence of SEQ ID NO:9 and the light chainconstant domain comprises the amino acid sequence of SEQ ID NO:10. 32.The isolated nucleic acid molecule of claim 28, wherein the light chainof the monoclonal antibody comprises the amino acid sequence of SEQ IDNO:17.
 33. The isolated nucleic acid molecule of claim 28, wherein thelight chain of the monoclonal antibody is kappa or lambda.
 34. Theisolated nucleic acid molecule of claim 28, wherein the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO:13. 35.The isolated nucleic acid molecule of claim 28, wherein the heavy chainconstant domain comprises the amino acid sequence of SEQ ID NO:8 oramino acid residues 1-326 of SEQ ID NO:8.
 36. The isolated nucleic acidmolecule of claim 28, wherein the heavy chain constant domain comprisesthe amino acid sequence of SEQ ID NO:11.
 37. The isolated nucleic acidmolecule of claim 28, wherein the isotype of the heavy chain is IgG. 38.The isolated nucleic acid molecule of claim 37, where the heavy chain isIgG1, IgG2, IgG3 or IgG4.
 39. An expression vector comprising thefollowing operably linked elements: a transcription promoter; a nucleicacid molecule encoding the light chain of the monoclonal antibody ofclaim 28; and a transcription terminator.
 40. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a nucleic acid molecule encoding the heavy chain of themonoclonal antibody of claim 28; and a transcription terminator.
 41. Arecombinant host cell comprising the expression vector of claim 40,wherein the cell expresses the heavy chain.
 42. The recombinant hostcell of claim 41, further comprising an expression vector comprising: atranscription promoter; a nucleic acid molecule encoding the light chainof the monoclonal antibody; and a transcription terminator; and whereinthe cell expresses the heavy chain and light chain.
 43. A method ofproducing a monoclonal antibody comprising: a) culturing the host cellof claim 42, wherein the expressed heavy and light chains form themonoclonal antibody; and b) recovering the monoclonal antibody.
 44. Anexpression vector comprising the following operably linked elements: atranscription promoter; a first nucleic acid molecule encoding the heavychain of the monoclonal antibody of claim 28; a second nucleic acidmolecule encoding the light chain of the monoclonal antibody of claim28; and a transcription terminator.
 45. A recombinant host cellcomprising the expression vector of claim 44, wherein the cell expressesthe heavy chain and light chain.
 46. A method of producing themonoclonal antibody encoded by the nucleic acid molecules in theexpression vector of claim 44 comprising: a) culturing a host cellcomprising said vector under conditions, wherein the encoded heavy andlight chains are expressed and form the monoclonal antibody; and b)recovering the monoclonal antibody.
 47. An expression vector comprisingthe following operably linked elements: a first transcription promoter;a first nucleic acid molecule encoding the heavy chain of the monoclonalantibody of claim 28; a first transcription terminator; a secondtranscription promoter; a second nucleic acid molecule encoding thelight chain of the monoclonal antibody of claim 28; and a secondtranscription terminator.
 48. A recombinant host cell comprising theexpression vector of claim 47, wherein the cell expresses the heavychain and light chain.
 49. A method of producing the monoclonal antibodyencoded by the nucleic acid molecules in the expression vector of claim47 comprising: a) culturing a host cell comprising said vector underconditions, wherein the encoded heavy and light chains are expressed andform the monoclonal antibody; and b) recovering the monoclonal antibody.50. An isolated nucleic acid molecule encoding a monoclonal antibodythat specifically binds to IL-23p19 (SEQ ID NO:6) and comprises a heavychain variable domain and a light chain variable domain, wherein theheavy chain variable domain comprises a CDR1 having the amino acidsequence of SEQ ID NO:19, a CDR2 having the amino acid sequence of SEQID NO:20, and a CDR3 having the amino acid sequence of SEQ ID NO:21, andwherein the light chain variable domain comprises a CDR1 having theamino acid sequence of SEQ ID NO:22, a CDR2 having the amino acidsequence of SEQ ID NO:23, and a CDR3 having the amino acid sequence ofSEQ ID NO:24.
 51. The isolated nucleic acid molecule of claim 50,wherein the light chain variable domain of the monoclonal antibodycomprises the amino acid sequence of SEQ ID NO:9.
 52. The isolatednucleic acid molecule of claim 50, wherein the light chain constantdomain of the monoclonal antibody comprises the amino acid sequence ofSEQ ID NO:10.
 53. The isolated nucleic acid molecule of claim 50,wherein the light chain variable domain of the monoclonal antibodycomprises the amino acid sequence of SEQ ID NO:9 and the light chainconstant domain comprises the amino acid sequence of SEQ ID NO:10. 54.The isolated nucleic acid molecule of claim 50 wherein the light chainof the monoclonal antibody comprises the amino acid sequence of SEQ IDNO:17.
 55. The isolated nucleic acid molecule of claim 50, wherein thelight chain of the monoclonal antibody is kappa or lambda.
 56. Theisolated nucleic acid molecule of claim 50, wherein the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO:7. 57.The isolated nucleic acid molecule of claim 50, wherein the heavy chainconstant domain comprises the amino acid sequence of SEQ ID NO:8 oramino acid residues 1-326 of SEQ ID NO:8.
 58. The isolated nucleic acidmolecule of claim 50, wherein the heavy chain constant domain comprisesthe amino acid sequence of SEQ ID NO:11.
 59. The isolated nucleic acidmolecule of claim 50, wherein the isotype of the heavy chain is IgG. 60.The isolated nucleic acid molecule of claim 59, where the heavy chain isIgG1, IgG2, IgG3 or IgG4.
 61. An expression vector comprising thefollowing operably linked elements: a transcription promoter; a nucleicacid molecule encoding the light chain of the monoclonal antibody ofclaim 50; and a transcription terminator.
 62. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a nucleic acid molecule encoding the heavy chain of themonoclonal antibody of claim 50; and a transcription terminator.
 63. Arecombinant host cell comprising the expression vector of claim 62,wherein the cell expresses the heavy chain.
 64. The recombinant hostcell of claim 63, further comprising an expression vector comprising: atranscription promoter; a nucleic acid molecule encoding the light chainof monoclonal antibody; and a transcription terminator; and wherein thecell expresses the heavy chain and light chain.
 65. A method ofproducing a monoclonal antibody comprising: a) culturing the host cellof claim 64, wherein the expressed heavy and light chains form themonoclonal antibody; and b) recovering the monoclonal antibody.
 66. Anexpression vector comprising the following operably linked elements: atranscription promoter; a first nucleic acid molecule encoding the heavychain of the monoclonal antibody of claim 50; a second nucleic acidmolecule encoding the light chain of the monoclonal antibody of claim50; and a transcription terminator.
 67. A recombinant host cellcomprising the expression vector of claim 66, wherein the cell expressesthe heavy chain and light chain.
 68. A method of producing themonoclonal antibody encoded by the nucleic acid molecule in theexpression vector of claim 66 comprising: a) culturing a host cellcomprising said vector under conditions wherein the encoded heavy andlight chains are expressed and form the monoclonal antibody; and b)recovering the monoclonal antibody.
 69. An expression vector comprisingthe following operably linked elements: a first transcription promoter;a first nucleic acid molecule encoding the heavy chain of the monoclonalantibody of claim 50; a first transcription terminator; a secondtranscription promoter; a second nucleic acid molecule encoding thelight chain of the monoclonal antibody of claim 50; and a secondtranscription terminator.
 70. A recombinant host cell comprising theexpression vector of claim 69, wherein the cell expresses the heavychain and light chain.
 71. A method of producing the monoclonal antibodyencoded by the nucleic acid molecule in the expression vector of claim69 comprising: a) culturing a host cell comprising said vector underconditions wherein the encoded heavy and light chains are expressed andform the monoclonal antibody; and b) recovering the monoclonal antibody.